extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
Impact of hydroxycarbamide and interferon-α on red cell adhesion and membrane protein expression in polycythemia vera

Polycythemia vera is a chronic myeloproliferative neoplasm characterized by the JAK2V617F mutation, elevated blood cell counts and a high risk of thrombosis. Although the red cell lineage is primarily affected by JAK2V617F, the impact of mutated JAK2 on circulating red blood cells is poorly documented. Recently, we showed that in polycythemia vera, erythrocytes had abnormal expression of several proteins including Lu/BCAM adhesion molecule and proteins from the endoplasmic reticulum, mainly calreticulin and calnexin. Here we investigated the effects of hydroxycarbamide and interferon-α treatments on the expression of erythroid membrane proteins in a cohort of 53 patients. Surprisingly, while both drugs tended to normalize calreticulin expression, proteomics analysis showed that hydroxycarbamide deregulated the expression of 53 proteins in red cell ghosts, with overexpression and downregulation of 37 and 16 proteins, respectively. Within over-expressed proteins, hydroxycarbamide was found to enhance the expression of adhesion molecules such as Lu/BCAM and CD147, while interferon-α did not. In addition, we found that hydroxycarbamide increased Lu/BCAM phosphorylation and exacerbated red cell adhesion to its ligand laminin. Our study reveals unexpected adverse effects of hydroxycarbamide on red cell physiology in polycythemia vera and provides new insights into the effects of this molecule on gene regulation and protein recycling or maturation during erythroid differentiation. Furthermore, our study shows deregulation of Lu/BCAM and CD147 that are two ubiquitously expressed proteins linked to progression of solid tumors, paving the way for future studies to address the role of hydroxycarbamide in tissues other than blood cells in myeloproliferative neoplasms.

P olycythemia vera is a chronic myeloproliferative neoplasm characterized by the JAK2V617F mutation, elevated blood cell counts and a high risk of thrombosis. Although the red cell lineage is primarily affected by JAK2V617F, the impact of mutated JAK2 on circulating red blood cells is poorly documented. Recently, we showed that in polycythemia vera, erythrocytes had abnormal expression of several proteins including Lu/BCAM adhesion molecule and proteins from the endoplasmic reticulum, mainly calreticulin and calnexin. Here we investigated the effects of hydroxycarbamide and interferon-α treatments on the expression of erythroid membrane proteins in a cohort of 53 patients. Surprisingly, while both drugs tended to normalize calreticulin expression, proteomics analysis showed that hydroxycarbamide deregulated the expression of 53 proteins in red cell ghosts, with overexpression and downregulation of 37 and 16 proteins, respectively. Within overexpressed proteins, hydroxycarbamide was found to enhance the expression of adhesion molecules such as Lu/BCAM and CD147, while interferon-α did not. In addition, we found that hydroxycarbamide increased Lu/BCAM phosphorylation and exacerbated red cell adhesion to its ligand laminin. Our study reveals unexpected adverse effects of hydroxycarbamide on red cell physiology in polycythemia vera and provides new insights into the effects of this molecule on gene regulation and protein recycling or maturation during erythroid differentiation. Furthermore, our study shows deregulation of Lu/BCAM and CD147 that are two ubiquitously expressed proteins linked to progression of solid tumors, paving the way for future studies to address the role of hydroxycarbamide in tissues other than blood cells in myeloproliferative neoplasms.
by thrombo-hemorrhagic complications and a propensity to transform into myelofibrosis and acute leukemia. 5 Reducing the vascular risk and preventing transformation are the main purpose of PV treatment. According to current guidelines, low-risk PV patients are managed with phlebotomy and aspirin, whereas high-risk patients receive cytoreduction with hydroxycarbamide (HC) or interferonα (IFN). 6 New therapeutic approaches aim at developing small molecules as specific inhibitors of JAK2 mutated forms. 7 HC, also known as hydroxyurea, is commonly used to treat PV patients requiring cytoreductive therapy. HC is a cytostatic non-alkylating hydroxylated urea analog that inactivates ribonucleotide reductase, the enzyme that catalyses the conversion of ribonucleotides to deoxyribonucleotides during de novo DNA synthesis, 8 leading to the interruption of DNA synthesis and cell death at the Sphase. 9 HC does not target the malignant clone and therefore appears unable to eradicate it. The efficacy of HC in preventing thrombosis was suggested in several randomized clinical trials, 10 but is still not proven. HC alone or in combination with other therapies may increase the risk of acute leukemia in MPN. 11 Interferon-α is a non-leukemogenic drug that, in some cases, induces cytogenetic remissions or reversion from monoclonal to polyclonal patterns of hematopoiesis, and may specifically target the malignant clone. 12 Its widespread use was hampered by its parenteral administration, cost and toxicity. The development of pegylated forms (PEG-IFN-α) increased tolerance and efficacy in IFN-treated patients. 13 Increased thrombotic risk in PV patients is associated with high levels of hemoglobin, impaired rheology, and increased viscosity resulting from erythrocytosis. [14][15][16] Targeting a hematocrit below 45% seems to decrease the thrombotic risk of these patients. 10 An additional parameter that might contribute to this risk was brought to light by our work showing abnormal activation of adhesion proteins in PV red blood cells (RBC). 17,18 More recently, using a proteomic approach, we also found that membranes of PV RBCs had abnormal expression of several proteins including the adhesion protein Lu/BCAM (Lutheran/Basal Cell Adhesion Molecule) and proteins from the endoplasmic reticulum, such as calreticulin (Calr) and calnexin. 19 These findings indicate that JAK2V617F not only impacts cell proliferation, but also induces changes in the repertoire of erythroid proteins expressed in circulating RBCs, which might contribute to the circulatory complications described in PV.
In this study, we investigated the effects of HC and IFN treatments on the expression of membrane proteins in circulating RBCs of PV patients. We show that HC and IFN each have a different impact on the expression and function of erythroid membrane proteins. While both drugs seem to normalize Calr expression, HC (but not IFN) enhances the expression of several proteins, including Lu/BCAM and CD147 adhesion proteins, and further exacerbates RBC adhesion to laminin.

Patients and blood samples
The study obtained ethical approval from Comité de Protection des Personnes Ile de France VII (PP 14-035); blood samples from 53 PV patients, followed at Saint Louis Hospital, Paris, were obtained with informed consent. Patients are divided in 3 groups: 25 patients (16 men and 9 women; mean age: 63±14 years) were treated with phlebotomy in addition to low-dose aspirin; 20 patients (11 men and 9 women; mean age: 64±16 years) were treated with HC [mean dose 0.8 g/day (0.3-1.5g/day)]; 12 patients (9 men and 3 women; mean age: 50±13 years) were treated with PEG-IFN-α according to international and local guidelines 20 [mean dose 115 mg/week (45-180 mg/week)]. The patient groups are small, and not necessarily matched for age or sex, partially due to the treatment guidelines of IFN and HC. Four patients from the first category were treated with HC during the study; blood samples from these 4 patients were collected before and during HCtreatment, at least six months after starting the treatment. All HCtreated patients were compliant to the treatment as they had complete hematologic response and increased mean corpuscular volume (MCV). JAK2V617F mutation was observed in all patients. Blood samples from 16 regular healthy donors at the Etablissement Français du Sang were also analyzed in this study (9 women and 7 men; mean age: 40±12 years, range 25-67 years). The 2 control (CT) blood samples used for the proteomic analysis, CT1 and CT2, were chosen to match as well as possible with the PV patient group of the study; they were from 67 and 57 year-old donors, respectively.

Blood samples and ghost preparation
Blood samples were collected on sodium heparin tubes. Buffy coat was removed and RBCs were cryopreserved at Centre National de Référence pour les Groupes Sanguins, Paris. RBC membranes were prepared by hypotonic lysis and washed with 5 mM sodium phosphate, pH 8.0 containing 0.2 mM phenylmethylsulfonylfluoride.

ITRAQ labeling and quantification by nano-liquid chromatography and mass spectrometry
Isobaric tag for relative and absolute quantitation (ITRAQ) multiplex analysis was carried out at the 3P5 proteomics facility as previously described, 21 with some modifications (Online Supplementary File 1). Trypsin digested peptides from red cell ghosts of 3 PV patients (before and during HC treatment: total 6 samples) and 2 healthy donors (CT) were processed. (For further details see Online Supplementary File 1). Data were analyzed using Protein Pilot 4 with the Uniprot human database. The analysis yielded 12,459 peptide spectrum matches corresponding to 2664 non-redundant peptides assigned to 375 proteins. Proteins were considered over-expressed or down-regulated when they showed PV/CT or PVHC/PV ratios ≥1.3 or ≤0.7, respectively, in at least 2 out of 3 PV samples, with no ratios lower than 1.2 or higher than 0.8 for over-expressed or down-regulated proteins, respectively.

JAK2V617F allele burden quantification
The percentage of the JAK2V617F allele was determined in DNA extracted from whole blood using the Mutaquant® (Ipsogen) method as described. 22

Flow adhesion assays
Red blood cell adhesion to laminin 521 was measured under flow conditions using a capillary flow chamber. Recombinant laminin 521 (BioLamina) at 5 ng/mL was immobilized in Vena8 Endothelial+ TM biochips (internal channel dimensions: length 20 mm, width 0.8 mm, height 0.12 mm). RBCs were perfused at 5.10 7 RBCs/mL for 5 minutes (min) at 0.5 dyn/cm² and 5-min washouts were performed at 0.5, 1, 2, 3, 5, 7 dyn/cm² using the ExiGo™ pump (Cellix Ltd., Dublin, Republic of Ireland). After each wash, adherent RBCs were counted in 6 representative areas along the centerline of the biochip using the AxioObserver Z1 microscope (10X objective) and AxioVision 4 analysis software (Carl Zeiss). Images of the same 6 areas were obtained throughout each experiment using the "Mark and Find" module of AxioVision analysis software.

Altered membrane protein expression in RBCs of HC-treated patients
In our recent work, we showed abnormal expression of several proteins at the membrane of PV RBCs, with a high number of proteins from the endoplasmic reticulum, including calreticulin (Calr). 19 To verify if HC treatment restores normal expression of these proteins, we performed a proteomic analysis with RBC ghosts from 3 patients before and during their treatment with HC (prepost patients). Three hundred and seventy-five proteins were confidently identified, with a number of peptides that allowed 358 distinct protein quantification (Online Supplementary File 2). Among the deregulated proteins initially reported, 19 HC decreased the expression of several over-expressed proteins and increased the expression of those down-regulated (Table 1). Nevertheless, the treatment did not seem to restore a normal expression pattern of these proteins for all 3 patients when compared to control (Table 1 and Online Supplementary Table S1). In addition, the comparative analysis showed that HC deregulated the expression of 53 proteins that had normal expression before the treatment started, with overexpression of haematologica | 2018; 103(6)  Table 2). Expression of Calr was diminished during HC treatment but did not reach control levels (Table 1). We analyzed Calr by western blot in RBC ghosts from a total of 4 prepost patients, including the 3 patients of the proteomics study, and found that its expression was decreased during the treatment without reaching significance ( Figure 1A), confirming the proteomics results. This was probably due to the variability and relatively short duration of the treatment in this group (mean duration 1.4 years, range 0.5-3 years). Therefore, we tested for the presence and expression level of Calr by western blot in a group of 11 patients treated with HC for a longer period of time (mean duration 5.9 years, range 0.5-20 years), a group of 19 untreated (UT) patients (i.e. with no cytotoxic antiproliferative treatment), and a group of 11 healthy donors ( Figure 1B). We found no significant difference between the HC and healthy donor groups indicating that long-term HC treatment restores the expression of Calr. Similarly, we investigated Calr in a group of 7 patients treated with IFN and found no difference with the healthy donors group, strongly suggesting that IFN restores normal expression of Calr in PV RBCs ( Figure 1B).

Overexpression of Lu/BCAM and CD147 in HC-treated patients
In the group of membrane proteins abnormally expressed in PV RBCs, 19 Lu/BCAM was the only protein whose overexpression was further exacerbated by HC in all 3 patients, with a fold increase comprised between 6.6 and 11.8 (Table 1). Lu/BCAM is a low abundance surface protein that is expressed on a subpopulation of circulating RBCs. To determine whether HC increases the percentage of Lu/BCAM-positive RBCs or Lu/BCAM expression level per RBC, or both, we performed flow cytometry assays with 18 UT and 17 HC blood samples, using a specific mouse monoclonal anti-Lu/BCAM antibody. The percentage of Lu/BCAM-expressing cells and the number of Lu/BCAM molecules per RBC, estimated by the mean fluorescence intensity (MFI) of Lu/BCAM-positive RBCs, were significantly higher in the HC group (median 94.35%, MFI=4570) than in the UT group (median 73%, MFI=1819) (P<0.001, Mann-Whitney test) (Figure 2A and B, and Online Supplementary Figure S1A). As Lu/BCAM expression is known to be higher in young (reticulocytes) than mature RBCs, 25 we determined the percentage of reticulocytes in all blood samples. There was no significant difference between both patient groups (mean UT:0.6 ±0.05; HC: 0.9±0.17, P=0.097) (Online Supplementary Figure  S1B) indicating that the increase of Lu/BCAM was not due to an HC-induced imbalance between reticulocytes and mature RBCs.
In order to explore the potential effect of HC and IFN on the expression of other erythroid adhesion proteins, we performed flow cytometry analysis of four additional adhesion markers: CD44, CD47, CD147 and CD242. All 4 markers are expressed on almost 100% of circulating RBCs and there was no effect of HC or IFN on this percentage (data not shown). Likewise, there was no effect of either HC or IFN treatment on the expression level per RBC of CD44, CD47 and CD242, estimated by the MFI,  Table 1. ITRAQ ratios of proteins with increased (i) or decreased (d) expression at the membrane of polycythemia vera (PV) red blood cells, and effect of hydroxycarbamide (HC) treatment. Proteins from endoplasmic reticulum are in bold.
when compared to UT patients. However, we observed increased expression of CD147 in the HC group (median MFI=8472) when compared to the UT group (median MFI=5406) (P<0.0001, Mann-Whitney test) ( Figure 2C) suggesting a common upregulation of Lu/BCAM and CD147 by HC. This increase was confirmed by proteomics in the 3 pre-post patients who showed increased CD147 expression during HC treatment (Table 2).

Increased RBC adhesion to laminin in HC-treated patients
As Lu/BCAM mediates abnormal adhesion of PV RBCs to laminin, 17 we examined the effect of HC treatment on PV RBC adhesion by performing adhesion assays under flow conditions. RBCs from HC-treated patients adhered much more than those from UT patients, with respective medians of 969 and 231 RBCs/mm 2 at 3 dyn/cm 2 (P=0.0047, Mann-Whitney test) whereas no significant difference was found between the IFN (271 RBCs/mm 2 ) and UT groups (P=0.8508, Mann-Whitney test) ( Figure  3A). Adhesion of RBCs from 6 control donors was minor (median=37 RBCs/mm 2 ), and significantly lower than in the UT, HC and IFN groups (P<0.01, Mann-Whitney test).
The impact of HC and IFN on cell adhesion was also analyzed by comparing the UT, HC and IFN groups in terms of RBC adhesion value distributions. We found that these values were broadly dispersed in the UT and HC groups but were less scattered in the IFN group ( Figure  3A), with a significant difference between the variance of the IFN group as compared to the UT (P<0.05) or the HC group (P<0.05) (Fisher exact test). This was probably linked to the JAK2V617F allele burden of each group. As a matter of fact, we have previously shown that PV RBC adhesion to laminin in UT patients was correlated with the JAK2V617F allele burden, defined as the percentage of circulating JAK2 alleles with the V617F mutation (%V617F). 26 We determined the %V617F of all patients and, as expected and as reported in previous studies, 13 the median was significantly lower in the IFN than in the UT and HC groups ( Figure 3B). Moreover, we found no significant difference between the UT and the HC groups, despite a lower median in the latter ( Figure 3B), indicating that increased RBC adhesion in the HC group was independent of the %V617F and was most probably due to the expression level of Lu/BCAM. This was demonstrated by plotting RBC adhesion values against the percentage of JAK2V617F ( Figure 3C) and the MFI of Lu/BCAM ( Figure  3D).

Longitudinal study of HC treatment in 4 PV patients
As only HC had activating effects on RBC adhesion, and because of wide interindividual variability in PV, we further investigated the effects of HC treatment in the 4 prepost HC patients of our cohort by performing adhesion and flow cytometry assays. This longitudinal analysis showed a great increase of adhesion after HC treatment for all 4 patients ( Figure 4A and B), confirming the results obtained with the UT and HC groups. As seen in the UT and HC groups, we found that the increased adhesion was associated with increased Lu/BCAM expression, both in haematologica | 2018; 103(6)  terms of percentage of Lu/BCAM-positive RBCs and associated mean fluorescence intensity ( Figure 4C), confirming that HC increases both the percentage of Lu/BCAM-positive RBCs and the expression of this protein within each positive RBC. This increase was reflected by higher amounts of Lu/BCAM during HC treatment detected by western blot (Figure 4D). We have shown that Lu/BCAM long isoform was constitutively phosphorylated in PV RBCs 18 and that inhibi-tion of this phosphorylation was associated with less PV RBC adhesion to laminin. 17 In order to determine whether the increased adhesion induced by HC was associated with Lu/BCAM activation, we tested the phosphorylation state of Lu/BCAM long isoform. Radiophospholabeling experiments confirmed that Lu/BCAM was phosphorylated in PV RBCs and showed increased phosphorylation during HC treatment ( Figure 4E, top panel). This increased phosphorylation was not only due to increased Lu/BCAM The top (P) and bottom (T) panels show the phosphorylation and the total amount of the immunopurified proteins, respectively. The phosphorylated fraction is determined by the P/T ratio.

A B
C D E expression, as the phosphorylation ratio (P/T), in which P is the measured phosphorylation and T the expression of total Lu/BCAM, was higher during than before HC treatment ( Figure 4E, bottom panel), indicating that HC both increases the expression of Lu/BCAM and the proportion of phosphorylated molecules.

Discussion
Our recent work showed abnormal expression of several proteins in RBCs of PV patients, 19 revealing qualitative alterations in these cells in addition to their increased number in the circulation. The aim of the current study was to address the effects of HC treatment on the expression of erythroid membrane proteins of PV patients using a global proteomic approach.
Our proteomic results obtained with ghosts from 3 prepost patients showed that HC treatment tends to restore the expression of deregulated membrane proteins. Nevertheless, normal expression of these membrane proteins was not reached, suggesting that the duration of the treatment for these patients was not sufficient, which is supported by the results of Calr expression in the group of 11 patients treated with HC for a longer duration.
Since we have shown that Calr overexpression was associated with the presence of JAK2V617F, 19 the decreased Calr expression in the HC group could have been the consequence of fewer JAK2V617F-positive clones in the bone marrow of these patients. This was reflected by their lower %V617F when compared to the UT group (median=42.5 and 60% for HC and UT, respectively), although the difference was not significant, which is in accordance with a study showing that HC does not appreciably reduce the JAK2V617F allele burden. 27 The IFN group also showed lower expression levels of Calr than the UT group, along with a significant decrease of the %V617F (median=15%). This decrease was expected as IFN treatment is known to reduce the JAK2V617F allele burden, with some cases of complete remission. 13 Calr is a calcium-binding chaperone that promotes efficient folding of glycoproteins but whose role in circulating RBCs has not been elucidated. Because calcium regulates several erythrocyte functions, 28 we believe that the decrease in Calr expression triggered by HC and IFN might be beneficial to the calcium homeostasis and subsequent calciumregulated functions of PV RBCs.
However, despite the tendency to normalize Calr expression, the proteomic analysis showed that HC treatment clearly affects the expression of several membrane proteins. This is not the first example of such deregulation, as HC is known to induce the neosynthesis of fetal hemoglobin in the erythroid lineage and is used to this purpose in sickle cell disease (SCD) patients. 29,30 Among the proteins over-expressed by HC in PV patients, Lu/BCAM and CD147 were also reported to be up-regulated in RBCs of sickle cell disease patients treated with HC, 31 indicating that the observed effect of HC is not specific to PV. This suggests the triggering of common pathways by HC independently of the underlying illness, most likely through the activation of gene transcription. This is supported by the findings of Odievre et al. 31 showing increased expression of Lu/BCAM in erythroid progenitors from SCD patients and healthy donors differentiated in vitro in the presence of HC. 31 Moreover, we have previ-ously shown activation of Lu/BCAM gene transcription by HC in endothelial cells grown in vitro. 32 Similar to our previous reports in sSCD, we found that HC significantly increases erythroid Lu/BCAM expression by enhancing both the percentage of Lu/BCAM-positive RBCs and the Lu/BCAM copy number per RBC. The increase in the percentage of Lu/BCAM-positive RBCs indicates that the observed higher expression is not due to the documented increase of the RBC volume consecutive to the HC treatment. Opposite to RBCs from HC-treated SCD patients, those from treated PV patients had increased adhesion to laminin. In SCD, increased Lu/BCAM phosphorylation is achieved in a PKA/cAMPdependent manner, with patients showing high cAMP levels that are decreased during HC treatment. 23 In PV, Lu/BCAM phosphorylation is driven by a JAK2V617Fdependent pathway involving Akt, and not PKA, which could explain the difference of HC effects between SCD and PV. HC is a nitric oxide (NO) donor in vivo [33][34][35] and its effect in PV RBCs might be due to Akt activation through an NO-dependent pathway. Indeed, RBCs have been shown to express an active NO synthase (NOS) 36 and are thus capable of generating NO and activating downstream effectors like Akt. Such activation of Akt by NO donors has been reported in endothelial cells 37 and chick retinal neurons; 38 it occurs through the activation of soluble guanylyl cyclase (sGC) and protein kinase G. Interestingly, HC has also been shown to activate sGC in erythroid cells, 39 such activation might trigger Akt activity in PV RBCs and lead to Lu/BCAM activation and increased RBC adhesion.
Activation of leukocytes, platelets and endothelial cells is known to promote a prothrombotic state. Markers for such activation have been reported in PV patients, including activation of polymorphonuclear leukocytes and endothelial cells, 40 and increased levels of leukocyteplatelet aggregates. 41 A previous report showed that treatment of PV patients with HC did not influence these marker levels. 42 Our study reveals a new pro-adhesive effect of HC in PV patients that acts by increasing the expression of two adhesion molecules, Lu/BCAM and CD147, and by activating RBC adhesion to laminin. CD147, also named basigin or neurothelin, is a member of the immunoglobulin superfamily that is expressed in various tissues, including brain, leukocytes, endothelial cells, and most tumor cell lines. CD147 is expressed during erythroid differentiation 43 and is carrier molecule for the blood group antigen Ok a . 44 Its erythroid adhesive function is important during the circulation of RBCs in the spleen. 45 It was shown to bind endothelial cells and fibroblasts, 46 and to be a receptor essential for erythrocyte invasion by Plasmodium falciparum. 47 It is noteworthy that Lu/BCAM and CD147 are adhesion markers linked to progression of solid tumors. Lu/BCAM is over-expressed in carcinomas in vivo and up-regulated following malignant transformation in some cell types. [48][49][50][51] Likewise, CD147 plays a central role in the progression of many cancers by inducing the secretion of matrix metalloproteinases and various cytokines (reviewed by Xiong, Edwards and Zhou 52 ). Several studies have indicated that CD147 is a multifunctional glycoprotein that inhibits tumor cell anoikis, 53 enhances tumor angiogenesis, 54 and promotes invasion, metastasis, 55 and glycolytic energy metabolism. 56 Considering the wide tissue distribution of both proteins, and the fact that HC distributes throughout the body reaching approximately all Effects of HC and IFN-α on PV RBC adhesion haematologica | 2018; 103 (6) tissues, one would expect that Lu/BCAM and CD147 would be also over-expressed in cell types other than RBCs, such as endothelial and epithelial cells. This is supported by our study showing that HC increases the endogenous expression of Lu/BCAM in endothelial cells ex vivo. 32 Consequently, overexpression of Lu/BCAM and CD147 in HC-treated patients might possibly have a negative impact both in the vascular territory by promoting abnormal cellular interactions, and in the higher incidence of skin cancer reported in HC-treated PV patients. 57 Although HC and IFN inhibit the proliferation of progenitor cells, nothing was known about their impact on PV RBCs once they exit the bone marrow. Our study is the first to address the effects of these molecules on PV RBCs, and to show that HC and IFN have a different impact on RBC protein expression and adhesive function. Leukocytosis and high hematocrit are two parameters involved in thrombotic events in MPN patients, and are both decreased during HC treatment. 10 A PV Study Group non-randomized trial showed that HC was associated with a lower incidence of early thrombosis compared to a historical cohort treated with phlebotomy alone (6.6% vs. 14% at 2 years). 58 Nevertheless, thrombosis is not totally abrogated in HC-treated patients. Patients treated with IFN also encounter less thrombotic events, 59 but whether this happens more or less than those treated with HC is still unknown because there had been no clinical trials comparing the effects of both drugs in the same cohort of MPN patients. Such trials are currently ongoing and might show differences between HC and IFN regarding circulatory complications.
Altogether, our study shows that HC and IFN each have a different impact on RBCs and reveals unexpected adverse effects of HC on RBC physiology in PV. Our findings show that HC deregulates the expression of several proteins at the red cell membrane providing new insights into the effects of this molecule on gene regulation and protein recycling or maturation during erythroid differentiation. Furthermore, our study shows that HC increases the expression of two ubiquitously expressed proteins that are linked to progression of solid tumors. Investigating such overexpression in tissues other than blood cells will be of interest in MPNs.


extfieldsofstudy: Biology Medicine

text:
Analysis of mitochondrial DNA sequence and copy number variation across five high-altitude species and their low-altitude relatives

Abstract High-altitude inhospitable environments impose a formidable life challenge for the local animals. Training and exposure to high-altitude environments produce both distinct physiological and phenotypic characteristics. The mitochondrion, an organelle crucial for the energy production, plays an important role in hypoxia adaptation. In this study, we investigated the mitochondrial DNA (mtDNA) polymorphism and copy number variation between the population pairs from distinct altitudes across the multi-species. Higher mitochondrial DNA control region’s genetic diversity is conspicuous in high-altitude animals versus low-altitude relatives. We also found an accordant decrease of mtDNA copy number in most of the tissues from high-altitude animals. Compared to mammals, chickens have significantly distinct mitogenomic characteristics, and more significant changes in the skeletal muscle mtDNA copy number between high- and low-altitude individuals. Our study catches a snapshot of the biological similarities and differences in the mitochondrial high-altitude acclimation across the species.

Introduction
Hypobaric hypoxia is associated with the extreme environmental characteristics of high-altitude environments, which is much more than that of lowland environments at similar latitudes. This imposes strong selective pressure on high-altitude animal species, both native and migratory. Compared with low-altitude animals, these animals possess unique high-altitude physiological adaptations and evolutionarily derived complex genetic architectures (Grether 2005). Tremendous genomic analyses have identified a vast array of rapidly evolving genes, contributing to the phenotypic change in the high-altitude animals (Simonson et al. 2010;Qiu et al. 2012;Li et al. 2013;Lorenzo et al. 2014). Recent comparative genomic analyses have revealed convergence and divergence in the gene expression changes between the species while adapting to high-altitude environments (Tang et al. 2015;Tang et al. 2017). Mitochondria, having an indispensable role in the energy metabolism, being extremely sensitive to the different energy-related selective pressures, more than the nuclear genome, is a core feature of extreme high-altitude acclimatization (Shen et al. 2009;Murray and Horscroft 2016). Mitochondrial genetic and phenotypic variations for high-altitude adaptation have been reported in mammals, including, among others, the Tibetan yak, Tibetan sheep, Tibetan pig, and bar-headed goose Gu et al. 2012;Nabholz et al. 2013;Hodgkinson et al. 2014;Liu et al. 2016). In this study, focus had been laid on the mitochondrial genome and mitochondrial copy number in the population pairs from distinct altitudes within a particular species. An attempt has been made to catch a glimpse of the consistencies and differences in the mitochondrial signatures shaped by high-altitude adaptation across multi-species.

Materials and methods
The detailed information of the animals and geographical locations are shown in Figure 1(a). The liver, brain, heart, lung, and skeletal muscle (the longissimus dorsi in four mammals and the chest muscle in chicken) were collected and used to extract complete DNA using MicroElute Genomic DNA kit (Omega, GA). Mitochondrial DNA (mtDNA) sequences from ten breeds were sequenced by an ABI 3730 DNA sequencer (Applied Biosystems, Foster City, CA). The mtDNA sequences in this study (GenBank accession numbers: MG837547-MG837556) were assembled manually using the computer program DNASTAR. A neighbor-joining tree was constructed using the Kimura 2-parameter method with software MEGA 6.0 (Tamura et al. 2013). Sequences of mitochondrial DNA D-loop region were downloaded from the GenBank (Table 1) and were used to calculate the nucleotide diversity (p), using the DNAsp software (Librado and Rozas 2009).
Relative amounts of nuclear DNA and mtDNA were determined using quantitative RT-PCR as previously described (Miller et al. 2003). The mitochondrial genes ATP6, COX1, and ND1 were used to quantify mtDNA. The GCG, AGRT1, SCD, CSN2b, and AGRT1, was used as the single-copy nuclear gene for pig, cattle/yak, sheep, goat, and chicken, respectively. Twice the ratio of ATP6/COX1/ND1 to single-copy nuclear gene number reflected the relative mtDNA content. Primer sequences used for mtDNA sequencing and mtDNA copy number detection are provided in Supplementary materials (available at https://doi.org/10.6084/m9.figshare.6603149).

Characteristics and phylogenetic analysis of mitogenomes
The mitochondrial genome sequences of ten breeds, within five species (pig, cattle, sheep, goat, and chicken, from highversus low-altitude regimes) were determined. Mitogenomes were all $16.5 kb in length with heavy nucleotide biases toward A and T (accounting for 60%), except in chicken (Figure 1(b)), which showed a relatively balanced nucleotide composition. Additionally, based on phylogenetic analysis, mammals sort into bird (chicken), omnivore (pig), and ruminant (goat, sheep, and cattle/yak) clades (Figure 1(c)), agreeing with established phylogenetic relationships across these species (Nabholz et al. 2009;Nabholz et al. 2013).

Nucleotide diversity of the mtDNA D-loop region
Mitochondrial DNA genetic markers can partly reflect the population fluctuations associated with local adaptations, especially the relatively sensitive mitochondrial control region (D-loop region) (Hassanin et al. 2009;Cheng et al. 2011). Here, we determined the similarities and differences in nucleotide diversity within the mtDNA D-loop region in a large-scale population analysis between high-and low-altitude species (Table 1). We observed relatively higher genetic diversity in the mtDNA D-loop region of high-altitude animals, which corresponds to previous results in the Tibetan pig (Li M et al. 2016), Tibetan sheep (Niu et al. 2017), Tibetan goat (Deng et al. 2017), and yak . We attribute the phenomenon to geographically isolating environments, and different domestication histories between the high-and low-altitude animals. Stronger artificial selection in low-altitude populations may contribute to the genetic diversity attenuation. Additionally, we found that different species exhibit different fold changes in the genetic diversity between high-and low-altitude populations, ranging from 1.05 in sheep to 15.6 in cattle/yak. The high-level diversity change in cattle/yak may be attributed to their highly diverged relationship (split from each other before 1.05-1.50 million years ago) (Qiu et al. 2012;Bai 2015). Interestingly, we found a significantly greater change in the genetic diversity (4.68 fold change in chicken, compared with 1.29 in pig, 1.11 in goat, 1.05 in sheep) between the pairs compared in bird than so in mammal.

Detection of mtDNA copy number
The decrease in mtDNA copy number associated with local animals living on the Tibetan Plateau has been demonstrated to be a long-term adaptive change contributed to high-altitude hypoxia adaption. We first investigated the mtDNA copy number in the population pairs from distinct altitudes within each group of a specific tissue (heart, skeletal muscle, lung, and brain) (Figure 2). For most of these tissues, we found the mtDNA copy number of high-altitude animals were lower than low-altitude counterparts. The result is consistent with the previous observations of decreased mitochondrial content with a concurrent smaller production of reactive oxygen species (ROS) across the vertebrate animal tissues, both in vitro and in vivo, when exposed to chronic hypoxia (Zhang et al. 2008;Heather et al. 2012;Li Y et al. 2016;Niu et al. 2017;Jin et al. 2018). However, increase in mitochondrial density occur in the early phases of acclimatization in response to acute hypoxia, which increases the metabolic cost of oxygen at the expense of cell damage by harmful   Figure 2. mtDNA copy number analysis of four tissues across five species, and a heatmap of mtDNA copy number ratio between low-and high-altitude animals (n ¼ 3/group) in those four tissues. Student's t-test, Ã p < 0.05, ÃÃ p < 0.01. Colored entries represent the log 2 mtDNA copy number ratio between low-altitude and high-altitude animals. The significant value was regarded as 1.5.
ROS (Luo et al. 2011;McElroy and Chandel 2017). Moreover, we found the mtDNA copy number in highly aerobic brain (Larson et al. 2014) and heart tissues (Nakada et al. 2017) to exhibit higher copy number levels than in other tissues, meanwhile exhibiting a relatively greater change ratio of mtDNA copy number between low-and high-altitude animals across five species (Figure 2). The lung, as the primary respiratory organ, exhibited relatively blunting response to the altitude in mitochondrial content change across the five species, and the lowest copy number levels of the four tissues. Interestingly, we observed the greatest mtDNA copy number change in chicken skeletal muscle (Figure 2). This observation raises the point that birds might have distinct mitochondrial phenotypic acclimatization processes for highaltitude hypoxia compared with mammals. Consistent with this, a previous study found hundreds of genes related to aerobic metabolism were differentially expressed in skeletal muscle between high-and low-altitude sparrows (Cheviron et al. 2008). Other studies have demonstrated different mtDNA sequence features (Nabholz et al. 2009) and physiological adaptations to high altitudes (Scott et al. 2009;Scott 2011;Murray and Horscroft 2016) between birds and mammals.

Conclusions
In this study, we determined pairwise differences in mitochondrial DNA polymorphism and copy number between high-altitude populations and low-altitude counterparts for each species. These mitochondrial gene sequence and content observations consistently indicate that birds have distinctly different mitochondrial acclimation processes to cope with high-altitude hypoxia than mammals. Our study provides insight to the general and specific characteristics of mitochondrial phenotypic acclimatization and genetic adaptation to high-altitude hypoxia in birds and mammals.
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extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Chemistry

text:
Metabolomic Study to Determine the Mechanism Underlying the Effects of Sagittaria sagittifolia Polysaccharide on Isoniazid- and Rifampicin-Induced Hepatotoxicity in Mice

In this study, a non-targeted metabolic profiling method based on ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was used to characterize the plasma metabolic profile associated with the protective effects of the Sagittaria sagittifolia polysaccharide (SSP) on isoniazid (INH)—and rifampicin (RFP)-induced hepatotoxicity in mice. Fourteen potential biomarkers were identified from the plasma of SSP-treated mice. The protective effects of SSP on hepatotoxicity caused by the combination of INH and RFP (INH/RFP) were further elucidated by investigating the related metabolic pathways. INH/RFP was found to disrupt fatty acid metabolism, the tricarboxylic acid cycle, amino acid metabolism, taurine metabolism, and the ornithine cycle. The results of the metabolomics study showed that SSP provided protective effects against INH/RFP-induced liver injury by partially regulating perturbed metabolic pathways.

Introduction
Tuberculosis is a pandemic chronic infectious disease caused by Mycobacterium tuberculosis.
Each year, about 9 million people are diagnosed with tuberculosis worldwide, and about 2 million tuberculosis patients die [1]. Isoniazid (INH) and rifampicin (RFP) are the first-line antituberculosis drugs recommended by the World Health Organization. These two drugs are metabolized in the liver, and both INH and RFP (INH/RFP) cause hepatotoxicity [2]. More importantly, RFP increases the hepatotoxicity of INH in a combination therapy involving INH/RFP [3].
INH/RFP causes irreversible damage, including liver failure, cell necrosis, inflammation, and steatosis [4][5][6]. These effects are considered to be mainly associated with oxidative stress [7], since INH was found to induce apoptosis via oxidative stress and to prevent Nrf2 translocation to the nucleus, thereby preventing its cytoprotective effect [8].
The hepatotoxicity of INH is mediated by its curtailment of mitochondrial function. Oxidative stress can damage the inner membrane of mitochondria, leading to energy metabolism disorders of the tricarboxylic acid cycle. Furthermore, INH and its metabolites (e.g., hydrazine) can cause mitochondrial injury, which can lead to mitochondrial oxidative stress and impairment of energy homeostasis [9].
Plant extracts are known to have the ability to alleviate liver injury by suppressing oxidative stress and hepatocyte apoptosis [10]. Particularly, plant polysaccharides can reduce pathological liver tissue damage, oxidative stress, and inflammatory cells induced by INH/RFP [11,12]. Sagittaria sagittifolia L. (Alismataceae) is a perennial aquatic herbal plant also known as Jiandaocao and Yanweicao. It is mainly distributed in the Yangtze River Basin in China and in other countries such as India and Japan. It was recorded in the Compendium of Materia Medica as a species native to China and is used as medicine and food [13]. S. sagittifolia is highly nutritious, has a pleasing taste, and is inexpensive. The extract of S. sagittifolia is known to restrain INH/RFP-induced liver injury [14], and the main hepatoprotective component is S. sagittifolia polysaccharide (SSP) [15]. However, the protective mechanism of SSP remains unclear.
Metabolomics involves the measurement of small-molecule metabolite profiles and fluxes in biological matrices after genetic modification or exogenous challenges [16]. An ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)-based metabolomics approach offers a lower detection limit, better selectivity, and higher sensitivity. To extend our previous findings concerning the antioxidative function of SSP, in this study, we used UPLC-HRMS to analyze the metabolites in the plasma of a mouse model to elucidate the mechanism of hepatoprotection rendered by SSP against liver injury caused by INH/RFP.

Instruments and Reagents
HPLC-grade methanol and acetonitrile were purchased from Beijing Invoke Technology Co. Ltd. (Beijing, China). Ultrapure water was prepared using a Milli-Q water purification system (Millipore, Bedford, MA, USA). S. sagittifolia was collected from Kunming City, Yunnan Province, China. The specimens were identified as corms of S. sagittifolia L. in Alismataceae by Professor Xiuli Wang, School of Chinese Materia Medica, Beijing University of Chinese Medicine, China. S. sagittifolia is an accepted name in the plant list [17].
SSP was prepared using a previously reported procedure that was simplified as follows [15]. The tuber roots of S. sagittifolia were dried at 55 • C and powdered. Then, 20 g of the powder was extracted with 200 mL of boiling water three times. The polysaccharides in the filtrate were precipitated fractionally with alcohol, at which point the proteins were removed from the product by the Sevag method [18]. The polysaccharides were further purified using diethylaminoethyl ion exchange cellulose (DEAE-52). The content of polysaccharide in the tuber roots was measured by the phenol-sulfuric acid method [19] at a wavelength of 490 nm and polysaccharide conversion factor (F) of 1.82. The results showed that the polysaccharide content was 34.31%, which was sufficient for subsequent experiments.

Mouse Model and SSP Administration
All animal studies followed the relevant national legislation and local guidelines and were performed at the College of Traditional Chinese Medicine at Beijing University of Chinese Medicine. A total of 24 BALB/c mice (20 ± 2 g) were commercially obtained from the Military Medical Science Academy of the PLA Animal Center (Beijing, China). Animals were caged under the supply of filtered pathogen-free air and provided food and water ad libitum for 7 days. The mice were randomly divided into three groups: control group (6 mice), model group (INH/RFP, 10 mice), and treatment group  (INH/RFP + SSP, 8 mice). The treatment group received SSP at a daily dose of 0.8 g/kg body weight by gavage; the control and model groups received the same amount of normal saline by gavage. After 4 h, the model and treatment groups received INH/RFP at a dose of (0.1 + 0.1) g/kg body weight by gavage. The control group was administered the same amount of normal saline by gavage. All groups received intragastric administration once per day for 30 days.

Plasma Sample Preparation
Samples were obtained from all animals 12 h after administration of the last dose; their body weight was measured, and blood was obtained from the eyeball. A portion of the plasma was used to detect the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) content. The remaining plasma was used for the detection of metabolites.
About 150 µL serum and 450 µL acetonitrile were vigorously shaken for 0.5 min, and the mixture was centrifuged at 15,700× g (13,000 rpm) for 10 min at 4 • C to precipitate the protein; the supernatant was re-centrifuged under the same conditions. The supernatant was then dried under nitrogen and dissolved in 160 µL mobile phase A (acetonitrile), followed by centrifugation at 15,700× g (13,000 rpm) for 10 min at 4 • C. The supernatant was then injected into a sample bottle.

UPLC-HRMS Analysis
For this analysis, the LTQ-Orbitrap XL mass spectrometer and Dionex UltiMate 3000 UHPLC Plus Focused system (Thermo Fisher Scientific, Waltham, MA, USA) were used. Chromatographic separation was performed on an ACQUITY UPLC TM BEH C18 column (2.1 × 100 mm, 1.7 µm; Waters, Milford, MA, USA) maintained at 30 • C. The mobile phase consisted of 0.1% acetonitrile (A) and ultrapure water (B). The elution gradient started at 5% A and increased to 35% at 0-3 min and then to 95% B at 3-8 min, followed by strong elution for 2 min and equilibration for 5 min. The flow rate was set to 0.34 mL/min, and the injection volume was 5 µL. An electrospray ionization source was used in both the positive and negative modes. The optimized conditions were as follows: capillary voltage, 35 V; drying gas flow, 11 L/min; gas temperature, 350 • C. The data were acquired using Fourier transform at high-resolution full scan mode (resolution, 30,000), and MS/MS spectra were collected using data-dependent acquisition.

Histological Analysis
Liver tissue was placed in 10% neutral formalin solution, embedded in paraffin wax, and cut into sections for hematoxylin and eosin (HE) staining. The histological activity scoring criteria of HE staining were as follows. Both lobular degeneration and portal inflammation were scored. Each item was assigned 0 points (no lesion), 1 point (the lesion area was less than 1/3 of the area of hepatic lobule and portal area), 3 points (the lesion area was less than 2/3 of the area of hepatic lobule and portal area, which was more than 1/3), or 4 points (the lesion range was more than 2/3 of the area of hepatic lobule and portal area).

Data Processing
The data obtained using UPLC-HRMS were imported to Sieve 2.1 software (Waltham, MA, USA) and then preprocessed for ion peak identification, screening, peak alignment, and noise filtering. The data matrices obtained were normalized. The pretreated data were analyzed using SIMCA-P13.0 (Umetrics, Umeå, Sweden), principal component analysis (PCA), and orthogonal signal correction, followed by partial least squares (PLS) mehod. The differential metabolites were identified using Compound Discoverer 2.0 (Thermo Scientific, Waltham, MA, USA) and compared with Human Metabolome Database (HMDB). Functional and pathway enrichment analyses using Kyoto Encyclopedia of Genes and Genomes (KEGG) were conducted to determine differential metabolic pathways [20]. Compound Discoverer 2.0 software (Thermo Scientific, Waltham, MA, USA) accurately matches mzCloud spectra of MS1 and MS2 mass spectral data with the appropriate molecular weight. The parameters were MS1 mass accuracy <5 ppm and a score matching threshold greater than or equal to 60 (the higher the matching, the closer the accuracy to 100). The library search algorithm was High Chem Low + High Res. During the search process, isotope matching and deduction of background noise (S/N ratio < 10,000 as cutoff) were performed.

Protective Effects of SSP on Ultrastructural Liver Damage Induced by INH/RFP
We performed histological activity score with inflammatory infiltration of hepatic lobule and portal area and both lobular degeneration and portal inflammation were scored. The result ( Table 1) indicated that the liver injury of the model group is significantly different from that of the control group and the treatment group.

Effects of SSP on ALT and AST Content
Compared with those in the control group, the ALT and AST levels increased significantly in the model group and decreased significantly in the treatment group ( Figure 1).
Molecules 2018, 23, x FOR PEER REVIEW 4 of 11 molecular weight. The parameters were MS1 mass accuracy <5 ppm and a score matching threshold greater than or equal to 60 (the higher the matching, the closer the accuracy to 100). The library search algorithm was High Chem Low + High Res. During the search process, isotope matching and deduction of background noise (S/N ratio < 10,000 as cutoff) were performed.

Protective Effects of SSP on Ultrastructural Liver Damage Induced by INH/RFP
We performed histological activity score with inflammatory infiltration of hepatic lobule and portal area and both lobular degeneration and portal inflammation were scored. The result (Table 1) indicated that the liver injury of the model group is significantly different from that of the control group and the treatment group.

Effects of SSP on ALT and AST Content
Compared with those in the control group, the ALT and AST levels increased significantly in the model group and decreased significantly in the treatment group ( Figure 1).

Normalization and Multivariate Statistical Analysis
Our metabolomics platform was used to analyze the statistically important metabolites. Unsupervised PCA was used first as an unbiased statistical method to investigate the general interrelation of groups, and a clear separation was obtained (Figure 2A,B). Next, PLS discriminant analysis (PLS-DA) was used to maximize the differences in metabolic profiles among the three groups, and the metabolites in the biological samples were detected ( Figure 2C,D). A good separation and a clear difference in blood metabolic profiles were noted among the groups, suggesting that INH/RFP and SSP might cause significant changes in the related components. The score plots from PLS-DA showed obvious separation among the three groups in both positive ( Figure 2E) and negative ion modes ( Figure 2F). The total values of R2Y and Q2, respectively, were higher than 0.6, suggesting that the PLS-DA model was reliable and stable and can sufficiently explain the differences between the two groups of samples. In addition, the model further confirmed that after SSP intervention, the metabolic components of the mice had obviously changed. The

Normalization and Multivariate Statistical Analysis
Our metabolomics platform was used to analyze the statistically important metabolites. Unsupervised PCA was used first as an unbiased statistical method to investigate the general interrelation of groups, and a clear separation was obtained (Figure 2A,B). Next, PLS discriminant analysis (PLS-DA) was used to maximize the differences in metabolic profiles among the three groups, and the metabolites in the biological samples were detected ( Figure 2C,D). A good separation and a clear difference in blood metabolic profiles were noted among the groups, suggesting that INH/RFP and SSP might cause significant changes in the related components. The score plots from PLS-DA showed obvious separation among the three groups in both positive ( Figure 2E) and negative ion modes ( Figure 2F). The total values of R2Y and Q2, respectively, were higher than 0.6, suggesting that the PLS-DA model was reliable and stable and can sufficiently explain the differences between the two groups of samples. In addition, the model further confirmed that after SSP intervention, the metabolic     . Metabolomic pathway analysis overview indicating selected metabolic pathways that were significantly affected among the control, model, and treatment groups. The size of each node indicates the pathway impact (based on the impact of each identified metabolite in a given pathway). The node color is graded depending on its p-value from pathway enrichment analysis (B). We used logarithmic analysis to produce the heat map, showing the 14 significantly altered metabolites based on ANOVA (C). Color from green to red represents the intensity of these metabolites from low to high levels in the analysis.

Discovery and Identification of Differential Metabolites
To analyze the metabolic data from these three groups in a biologically meaningful manner, enrichment and metabolite topological analyses of the identified metabolites were carried out. An overview of the pathway analysis is shown in Figure 3B. The pathway analysis contained all the matched pathways arranged by p-value on the Y-axis, based on the pathway enrichment analysis, and the pathway impact values arranged on the X-axis, based on the pathway topology analysis. Therefore, many metabolic pathways such as the citrate cycle, arginine, and proline metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis; and valine, leucine, and isoleucine biosynthesis were significantly affected. These findings demonstrate that more than 18 metabolic pathways were significantly dysregulated in the three groups. According to the variable importance in projection (VIP) of the PLS-DA model, the parameters for the evaluation of potential biomarkers were scored at VIP > 1 (the expressed sequence selection of these variables is higher than the average level of clustering effect). Next, t-test and compound screening showed marked differences between the metabolites obtained from the control and model groups. Variables showing differences at p < 0.05 were considered potential biomarkers, and 14 compounds that matched those included in the HMDB metabolite retrieval were finally screened. Table 2 showed the mass-charge ratio (M/Z), formula and related pathways of metabolites. These compounds were related to the metabolic pathways of fatty acid metabolism, amino acid metabolism, tricarboxylic acid cycle, and the ornithine cycle (Table 2). A heat map was generated to depict all the metabolites that were significantly altered in the dataset ( Figure 3C).

Metabolic Pathway Analysis
The potential metabolites shown in Table 2 are involved in various metabolic pathways. We investigated the links between these metabolites using the KEGG database and HMDB and generated a metabolic map (Figure 4). The map suggested that INH/RFP and SSP had an important influence on the energy metabolism of mice. To further evaluate the effect of pre-administration of SSP on the potential biomarkers, one-way ANOVA with Tukey's post hoc test was performed between the three groups by SPSS software. The relative peak intensity of the 14 metabolites is shown in Figure 4. Compared with those in the model group, nine metabolites including taurine, L-isoleucine, aspartic acid, ornithine, indole-5,6-quinone, succinic acid, adrenaline, palmitic acid, and L-histidine were significantly recovered in the treatment group. The other five metabolites were also reversed in the model group compared with the control group.  Figure 3. The S-plot obtained using positive and negative ion mode datasets of the control and model groups (A). Metabolomic pathway analysis overview indicating selected metabolic pathways that were significantly affected among the control, model, and treatment groups. The size of each node indicates the pathway impact (based on the impact of each identified metabolite in a given pathway). The node color is graded depending on its p-value from pathway enrichment analysis(B). We used logarithmic analysis to produce the heat map, showing the 14 significantly altered metabolites based on ANOVA (C). Color from green to red represents the intensity of these metabolites from low to high levels in the analysis.

Metabolic Pathway Analysis
The potential metabolites shown in Table 2 are involved in various metabolic pathways. We investigated the links between these metabolites using the KEGG database and HMDB and generated a metabolic map (Figure 4). The map suggested that INH/RFP and SSP had an important influence on the energy metabolism of mice. To further evaluate the effect of pre-administration of SSP on the potential biomarkers, one-way ANOVA with Tukey's post hoc test was performed between the three groups by SPSS software. The relative peak intensity of the 14 metabolites is shown in Figure 4. Compared with those in the model group, nine metabolites including taurine, L-isoleucine, aspartic acid, ornithine, indole-5,6-quinone, succinic acid, adrenaline, palmitic acid, and L-histidine were significantly recovered in the treatment group. The other five metabolites were also reversed in the model group compared with the control group. Figure 4. The influence of SSP and INH/RFP was associated with fatty acid metabolism, taurine and hypotaurine metabolism, amino acid metabolism, the tricarboxylic acid cycle, and the ornithine cycle. The different colors of the metabolites represent the following: red, increased in a given group; blue, decreased; and black, not detected. Bar plots show UPLC-HRMS relative signal intensities of the metabolites in the positive and negative modes in the control, model, and treatment groups. In each histogram, red represents the relative expression of the control group, green represents the model group, and blue represents the treatment group. Data are expressed as mean ± SD. * p < 0.05 versus control group; # p < 0.05 versus model group. Figure 4. The influence of SSP and INH/RFP was associated with fatty acid metabolism, taurine and hypotaurine metabolism, amino acid metabolism, the tricarboxylic acid cycle, and the ornithine cycle. The different colors of the metabolites represent the following: red, increased in a given group; blue, decreased; and black, not detected. Bar plots show UPLC-HRMS relative signal intensities of the metabolites in the positive and negative modes in the control, model, and treatment groups. In each histogram, red represents the relative expression of the control group, green represents the model group, and blue represents the treatment group. Data are expressed as mean ± SD. * p < 0.05 versus control group; # p < 0.05 versus model group.

Discussion
S. sagittifolia is recorded in ancient Chinese literature as having various effects, such as a warming and nourishing effects on the viscera [21]. Further, its leaf is used to treat insect and snake bites. When cooked, it can promote digestion and absorption and can be used to treat urethritis and beriberi. Modern research shows that S. sagittifolia can relieve liver injury caused by cadmium [22], CCl 4 [23], and INH/RFP [24]. The mechanism of action might be related to anti-lipid peroxidation and enhancement of free radical scavenging activity [14]. In our study, SSP was also found to protect against liver injury caused by INH/RFP (Table 1).
In this study, UPLC-HRMS was used to investigate the plasma metabolic profile of a mouse model and subsequently elucidate the effect of SSP on liver injury. Fourteen potential biomarkers were identified; these were related to disturbances of the metabolism primarily involving fatty acid metabolism, taurine metabolism, the tricarboxylic acid cycle, ornithine cycle, and amino acid metabolism. Among them, amino acid metabolism is very important because the liver is the only organ that can metabolize all amino acids. Based on the current data, the levels of branched-chain amino acids (BCAAs), histidine, methionine, phenylalanine, and tryptophan (aromatic amino acids) were significantly changed. In the model group, BCAAs were downregulated, whereas the other amino acids were upregulated. This finding is consistent with that of a previous study [25]. In the model group, the pathway through which phenylalanine is broken down into fumaric acid and epinephrine was inhibited. In a previous study, phenylalanine hydroxylase activity was inhibited in liver injury [26]. Therefore, the protective effects of SSP on liver injury might be related to enhancement of the metabolism of phenylalanine.
Furthermore, liver damage often leads to changes in amino acid metabolism. Alterations in amino acid metabolism associated with liver disease are characterized by low levels of circulating BCAAs and elevated levels of circulating aromatic amino acids (phenylalanine, tryptophan, and tyrosine) and methionine [27]. BCAAs can inhibit aromatic amino acids (AAAs) crossing the blood-brain barrier, reduce high ammonia levels in the blood, and maintain the nitrogen balance in the body. BCAAs have important functions such as protection against liver injury. Dietary supplementation of BCAAs ameliorated liver fibrosis in a diethyl nitrosamine-induced rat model of hepatocellular carcinoma with liver cirrhosis [28]. In addition, BCAAs can directly exacerbate hepatic lipotoxicity by reducing lipogenesis and inhibiting autophagy in hepatocytes [29]. Leucine was found to reduce fat synthesis in mouse liver fat cells [30]. In this study, BCAAs were downregulated, while AAAs were upregulated in the model group, indicating that liver injury induced by INH/RFP resulted in a decrease in the clearance of phenylalanine and the release of a large amount of phenylalanine into the blood. Thus, SSP could protect the liver by regulating the metabolism of BCAAs.
Taurine is the most abundant free amino acid in animals and has a wide range of biological functions. For example, it can scavenge oxygen free radicals and resist lipid peroxidation. The liver is not only the main site of taurine synthesis but also an important target organ of taurine. Taurine synthesis decreases when inflammation and liver necrosis occur. It is not only a potential marker of liver injury but also a potential therapeutic agent for liver injury. In the liver, taurine binds to bile acids and participates in bile excretion. Taurine has a protective effect against liver injury related to its role in reducing steatosis and lipid peroxidation [31]. In addition, taurine protects isolated hepatocytes against CCl 4 and hydrazine cytotoxicity; the mechanisms may include the modulation of calcium levels, osmoregulation, and membrane stabilization [32]. In this study, the content of taurine increased in the model group and decreased in the treatment group, indicating that liver injury caused by INH/RFP resulted in a decrease in taurine synthesis, and the decrease resulted in cholestasis. Therefore, SSP could reduce liver damage by regulating taurine synthesis.
The liver plays an important role in the metabolism of fatty acids since it takes up a large proportion of free fatty acids (FFAs) entering the splanchnic bed through the portal vein [33], with only a small fraction of FFAs being taken up by the non-hepatic splanchnic bed. When the metabolism of liver fatty acids is insufficient, the content of FFAs in the blood and their uptake by the liver increase; this might lead to the accumulation of lipids in liver cells, causing cytotoxicity. Under normal circumstances, mitochondria can decompose excessive amounts of fatty acids in cells via fatty acid beta-oxidation and produce ATP, although the electron respiratory chain leads to excessive generation of reactive oxygen species (ROS) [34]. Overproduction of ROS results in damage of the mitochondrial structure and function and thus the function of the electron respiratory chain, further increasing cellular oxidative stress and causing impaired fatty acid catabolism. As a result, fatty acids are deposited in the cytoplasm [35]. Palmitic acid is an important component of FFAs in the blood. However, the deposition of fatty acids causes cellular toxicity. Palmitic acid has been shown to exhibit a dose-dependent cytotoxic effect associated with ROS production, increasing the level of markers associated with apoptosis and necrosis and decreasing albumin production [36]. Some studies suggested that palmitic acid promotes apoptosis by inducing autophagy in hepatocytes [37]. In this study, the content of palmitic acid was higher in the model group, suggesting that INH/RFP caused lipid deposition in the model group mice, which is somewhat evident in Table 1. Hence, palmitic acid might be a potential biomarker of hepatic damage caused by INH/RFP. Liver cell damage caused by liver disease can affect the elimination of blood ammonia [38], causing high blood ammonia levels and further liver damage [39]; therefore, a vicious cycle is formed. Under normal circumstances, the liver can metabolize ammonia, a toxin that can decrease the level of ATP in the brain and in turn affect nerve function and nerve cell metabolism, via the ornithine cycle to produce urea, which is then excreted through the kidneys. In this study, the level of ornithine and aspartic acid was higher in the treatment group, indicating that the metabolites of ammonia from the ornithine cycle were higher. Thus, SSP might alleviate liver injury by maintaining the function of the ornithine cycle, and ornithine and aspartic acid might also be potential biomarkers of hepatic damage caused by INH/RFP. Some studies found that INH/RFP caused liver oxidative stress [40,41], which increased superoxide dismutase activity. Subsequently, peroxidative damage of the mitochondrial endometrium was induced, which activated respiratory chain complexes, leading to a decrease in both ATP synthase and oxidative phosphorylation function. These events culminated in reduced ATP yield and energy metabolism disorders of the tricarboxylic acid cycle [42]. The tricarboxylic acid cycle is a metabolic process that occurs in mitochondria and produces ATP to provide energy for the body. Succinic acid and fumaric acid are important intermediates of the tricarboxylic acid cycle, and their expression can be influenced by liver injury. In this study, succinic acid and fumaric acid were downregulated in the model group and upregulated in the treatment group, suggesting that SSP may protect the tricarboxylic acid cycle and thus prevent liver damage caused by INH/RFP.

Conclusions
In summary, INH/RFP was found to disrupt metabolism related to energy demand. The PLS-DA-mediated classification for the selection and validation of biomarkers revealed 14 metabolites that could be considered as potential biomarkers. The UPLC-HRMS-based metabolomics method was successfully applied to evaluate the protective effect of SSP. When the potential biomarkers were used as screening indexes, SSP was found to restore fatty acid metabolism, taurine and hypotaurine metabolism, amino acid metabolism, the tricarboxylic acid cycle, and the ornithine cycle. Therefore, SSP may be a promising agent to prevent hepatic injury induced by INH/RFP. However, further studies with a larger sample size are warranted to confirm our findings.


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Up-regulated autophagy: as a protective factor in adipose tissue of WOKW rats with metabolic syndrome

Background Wistar Ottawa Karlsburg W (RT1u) rats (WOKW) are a model of the metabolic syndrome (MetS). Adipose tissue (AT) and peripheral nerves of WOKW rats exhibit up-regulated autophagy and inflammation corresponding with decreased apoptosis rate. The aim of this study was to characterize AT in WOKW rats in relation to autophagic activity. Methods mRNA and protein expression of adiponectin, pro-inflammatory and pro-apoptotic markers including MCP1, TNFα, cleaved caspase-3 and RNF157, a new candidate gene regulated through autophagy, were analyzed in adipocytes isolated from visceral and subcutaneous AT of 5-month old WOKW rats with MetS and LEW.1W controls in response to pharmacological inhibition of autophagy. Immunohistochemistry was performed to detect adiponectin and RNF157 protein in cultured adipocytes. Results Inhibition of autophagy by LY294002 was associated with a fourfold up-regulation of adiponectin expression and a decrease of RNF157 protein and pro-inflammatory markers—MCP-1 and TNFα predominantly in visceral adipocytes of obese WOKW rats compared to LEW.1W rats. Moreover, inhibition of autophagic activity correlates with an activation of cleaved caspase-3 apoptotic signaling pathway. Conclusions Up-regulated autophagy in obese WOKW rats contributes to the regulation of visceral AT function and involves an altered balance between pro-inflammatory and protective adipokine expression. Our data suggest that activation of AT autophagy protects against adipocyte apoptosis at least under conditions of obesity related MetS in WOKW rats. Electronic supplementary material The online version of this article (10.1186/s13098-018-0317-6) contains supplementary material, which is available to authorized users.

Inflammation of adipose tissue (AT)-a symptom of AT dysfunction both in humans and model organisms which develops with weight gain and diabetes-is associated with AT autophagic activity [7][8][9]. Autophagy is an essential, lysosomal degradation process, which is implicated in cellular homeostasis through break down and recycling of aggregated proteins and damaged organelles [10]. However, activated autophagy has been generally considered as a cell-protection mechanism, which promotes cell survival; the excessive activation of autophagy may lead to autophagic cell death [11].
Autophagy has been shown to prevent or induce inflammatory responses and to regulate of cytokine production in AT of obesity in humans and animal models [12][13][14]. The molecular mechanisms underlying the interaction between altered immune-response and autophagy regulation in obesity are still poor investigated.
Recently, we have shown that enhanced autophagy characterized by up-regulated expression of autophagy genes in AT and peripheral nerves occurs in obese Wistar Ottawa Karlsburg W (RT1u) (WOKW) rats, a model for MetS [15,16]. WOKW rats with the MHC RT1u haplotype develop a polygenetically inherited metabolic syndrome with obesity, hypertension, dyslipidemia and hyperinsulinemia, closely reflecting the human MetS [17][18][19]. Up-regulation of autophagy in WOKW rats corresponded with macrophage infiltration in the sciatic nerves and AT without signs of neuropathy or diabetes. Moreover, activated autophagy seems to protect from caspase-3 mediated apoptosis in AT and nerves of WOKW rats [15,16]. There is strong evidence for a causal relationship between autophagy and AT function in obesity.
Here, we tested the hypothesis that autophagic activity contributes to the regulation of protective, inflammatory and apoptotic factors in adipocytes isolated from visceral and subcutaneous AT of WOKW rats. We provide data suggesting that AT autophagy represents a key mechanism protecting against the development of obesityrelated metabolic comorbidities.

Animals
Five-month old WOKW (n = 12) and LEW.1W (n = 12) male rats were bred and kept under standard conditions maintained at 21 ± 1 °C on a 12:12 h light/dark cycle (5 a.m./5 p.m.) at the Saxon Incubator of Clinical Translation (SIKT, Leipzig, Germany). All rats had free access to water and were fed with a regular chow food (Sniff, Soest, Germany). Body weight was recorded for each group (WOKW mean 673 g, p < 0.05; LEW.1W mean 503 g, p < 0.05).
Experiments followed the international guidelines of animal care and the study protocols were approved by the Landesdirektion Leipzig, the local authority for animal care (T01/13 and T08/16). All rats were sacrificed via an isoflurane overdose at the end of the experiment.

Phenotypic characterization
At an age of 5 months all animals were weighed, blood was collected and whole body composition (fat mass, lean mass and total body water) was determined in awake rats by using nuclear magnetic resonance technology with EchoMRI700 ™ instrument (Echo Medical Systems, Houston, TX, USA). Data were analyzed by the manufacturer's software.
Serum insulin concentrations were measured by ELISA using Mercodia Rat ELISA (Uppsala, Sweden). Blood glucose values were determined from whole venous blood samples using an automated glucose monitor (FreeStyle mini, Abbott GmbH, Ludwigshafen, Germany). HbA1c levels were analysed by an automatic chemical analyzer in our Institute of Laboratory Medicine and Clinical Chemistry.

Primary adipocyte cell cultures
WOKW and LEW.1W rats were anesthetized with 2.5% isoflurane inhalation. Subcutaneous and visceral adipose tissue depots were removed under sterile conditions, washed in PBS and incubated in a 5 ml of preadipocyte isolation buffer containing 123 mM NaCl, 5 mM KCl, 1.3 mM CaCl 2 , 5 mM Glucose, 100 mM HEPES, 4% BSA (bovine serum albumin fraction V) and 0.2% collagenase (type 1). Incubations were performed in a water bath with rotating flasks (120 rpm) at the temperature 37 °C for 40 min. After incubation the tissue remnants and cell aggregates were removed by filtration through a 100 µm nylon screen. The cells passing the filter were pelleted by centrifugation (5 min, 1500 rpm at RT) and then resuspended in an erythrocyte lysis buffer containing 0.154 M NH 4 Cl, 0.01 M KHCO 3 and 0.1 mM EDTA for 10 min at RT. After incubation the preadipocyte pellet was resuspended and maintained in high-glucose Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma) at 37 °C in a 5% CO 2 atmosphere. Approximately 20,000 cells per well of a 24-well plate or per 30-mm culture dishes were obtained.
At day 2 of confluence, preadipocytes were induced to differentiate into adipocytes by using medium with 1 mM insulin, 0.4 mg/ml dexamethasone and 0.5 mM 3-isobutyl-1-methylxanthine changed every 48 h. After additional 7 days, about 85% of the cells had accumulated large-sized fat droplets as verified by phase contrast microscopy. For studies of autophagy dependent regulation of adiponectin, MCP-1, TNFα, RNF157 and cleaved caspase-3 expression, adipocytes were used at day 9 after the induction of differentiation.

Inhibition of the PI3K pathway
Phosphorylation of phosphatidylinositol 3-kinase (PI3K) is required for autophagy activation, synthesis of autophagosomes and lipidation of cytosolic LC3-I into the active, autophagosome membrane-bound form, LC3-II. LY294002 is a specific inhibitor of PI3K phosphorylation [20].
Dose-dependent inhibitory effect of LY294002 on PI3K phosphorylation was studied by treating adipocyte cells with increasing concentrations of LY294002 (20,30 and 50 µM) in serum-free medium for 48 h. Completely inhibited phosphorylation was obtained at the concentration of 50 µM. To inhibit autophagy process, visceral and subcutaneous adipocytes were incubated in serumfree DMEM medium at 37 °C in the presence or the absence of LY294002 at a concentration of 50 µM for 48 h (Fig. 1a).

RT-PCR analyses
For mRNA analyses, subcutaneous and visceral AT depots of WOKW and LEW.1W rats (n = 4) were removed under 2.5% isoflurane anesthesia, transferred on ice, rinsed in PBS and immediately frozen in liquid nitrogen. mRNA expression of rat marker genes of Rnf157 and Mcp-1 was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using the TaqMan assay, and fluorescence was detected on an ABI-PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany). Total RNA was isolated using TRIzol (Life Technologies, Grand Island, NY). 1 μg RNA was reverse transcribed with standard reagents (Life Technologies). From each RT-PCR consisting of an initial denaturation at 95 °C for 10 min, followed by 40 PCR cycles, each cycle consisting of 95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min. 2 μl was amplified in a 20-μl PCR using the Brilliant SYBR Green PCR mix from Thermo Fisher Scientific (Waltham, MA, USA). The following primers were used: rat Rnf157, 5′-AACAGCCAAGGGCTCAAACT-3′ (sense) and 5′-TCTGACTCACTGCAAGAGCG-3′ (antisense); rat Mcp-1, 5′-TAGCATCCACGTGCTGTCTC-3′ (sense) and 5′-CAGCCGACTCATTGGGATCA-3′ (antisense) (Biomers, Ulm, Germany). Rat Rnf157 and Mcp-1 mRNA expression was calculated relative to the mRNA expression of 18S rRNA, determined by a premixed assay on demand for human 18S rRNA (Hs99999901_s1; Life Technologies, CA). Amplification of specific transcripts was confirmed by melting curve profiles (cooling the sample to 60 °C and heating slowly to 95 °C with measurement of fluorescence) at the end of each PCR. Specificity of PCR was verified by subjecting the amplification products to agarose gel electrophoresis, which verified the presence of a single band of appropriate molecular weight for each amplification product.

Statistical analyses
Data are presented as mean ± SEM (Figs. 1, 3, 4, 5, 7). Differences between the groups were validated by oneway-ANOVA and the Newman-Keuls test using SigmaStat (Jandel Scientific, San Rafael, CA). A value of p < 0.05 was considered statistically significant.

Phenotypic characterization of WOKW rats
The phenotype of the 5-months-old WOKW (n = 24) and LEW.1W control rats (n = 24) is summarized in Table 1. WOKW rats have a significantly higher body weight as well as fasting serum insulin concentrations compared to healthy LEW.1W rats. In contrast, HbA1c levels and fasted blood glucose did not differ between both strains. The adiposity index was significantly increased in WOKW compared to LEW.1W rats ( Table 1).

Inhibition of autophagy in visceral and subcutaneous adipocyte cell cultures of WOKW and LEW.1W rats
We have previously shown the strikingly up-regulated autophagy in adipose depots of obese WOKW rats with metabolic syndrome in correlation with inhibition of caspase-3 apoptotic signal pathway [15].
Here, we extent these findings by pharmacologically inhibiting autophagy in cultured adipocytes of obese WOKW rats with MetS and in lean LEW.1W control healthy rats. Mature visceral and subcutaneous adipocytes from both strains have been inhibited with an autophagy inhibitor-LY294002 at the concentration of 50 µM for 48 h.
As shown in Fig. 1, phosphorylation of key autophagy signaling pathway-PI3K p85 and Akt protein in visceral and subcutaneous adipocytes of WOKW and LEW.1W rats was inhibited with LY294002 inhibitor after 45 min of treatment. Next, we assessed the protein level of the autophagy related genes-Atg5/12, LC3 and mTOR in adipocyte cell cultures after inhibition.  10:13 Visceral adipocyte cell cultures of WOKW and LEW.1W rats display a significant decrease of LC3 immunoreactivity compared to the non-inhibited control cultures after inhibition with LY294002 for 48 h (Fig. 2a-d).
Western blot analyses confirmed an inhibitory effect of LY294002. Expression of Atg5/12 protein was decreased about threefold in visceral adipocytes of WOKW rats and 1.3-fold in LEW.1W rats (Fig. 3a, b). LC3-II (membrane associated, 16 kDa form of LC3) displayed a 2.6fold down-regulation in visceral adipocytes of WOKW rats and a 3.7-fold decrease in LEW.1W rats. In subcutaneous adipocytes, a 1.6-fold down-regulation of LC3 protein expression was found in WOKW rats and 4.5fold decrease in LEW.1W control rats (Fig. 3a, c). Expression of mTOR was increased about 1.6-fold in visceral adipocytes of both strains. Contrary, in subcutaneous adipocyte cell cultures only LEW.1W rats show higher expression of mTOR after autophagy inhibition (Fig. 3a,  d).

Autophagy dependent regulation of adiponectin, MCP-1 and TNFα protein expression in cultured adipocytes of WOKW and LEW.1W rats
In the obese state, the adipocytes increase secretion of pro-inflammatory chemokines and cytokines, such as MCP-1, TNFα or interleukins (ILs) which are involved in insulin resistance [21][22][23]. Moreover, the expression of the insulin-sensitizing adipokine-adiponectin is strikingly down-regulated in obesity [24]. AT protein level of adiponectin correlates with up-regulated autophagy and was lower in cultured adipocytes of obese WOKW rats as compared to LEW.1W control rats.
Moreover, we confirm the up-regulated mRNA (Additional file 1: Figure S1) and protein expression of MCP-1 in adipose depots of WOKW rats in comparison to LEW.1W control rats mainly in visceral adipocytes (Fig. 4a, c). MCP-1 protein expression was decreased in visceral adipocytes of both strains after LY294002 treatment. In contrast, MCP-1 protein in subcutaneous adipocytes is down-regulated in LEW.1W rats only (Fig. 4a,  c).
Additionally, we have found a fourfold increase in TNFα protein expression in visceral adipocyte cell culture of WOKW rats, which could be significantly attenuated after autophagy inhibition. The expression of TNFα in subcutaneous adipocytes has been detected at comparable levels between all experimental groups (Fig. 4a, d).
As shown in Fig. 5a-d immunohistofluorescence analysis of adiponectin distribution in visceral adipocyte cell cultures demonstrating an increase of adiponectin immunoreactivity after LY294002 treatment. Immunofluorescence of adiponectin in subcutaneous adipocytes was rarely detectable.

Regulation of RNF157 in adipocyte cell cultures in obese WOKW rats
Unexpectedly, a strikingly high mRNA expression of new candidate gene-Rnf157, a neuroprotective protein [25], has been detected in visceral and subcutaneous depots of WOKW rats and in visceral fat of LEW.1W rats (Fig. 6a-f ).
Interestingly, we found a 5-and 2.6-fold decrease of RNF157 protein expression after autophagy inhibition in visceral adipocyte cell cultures of WOKW and LEW.1W rats, respectively. Subcutaneous adipocytes of WOKW rats exhibit a 1.4-fold decrease in RNF157 expression after treatment. No changes in RNF157 expression were observed in subcutaneous adipocyte cultures of LEW.1W rats after autophagy inhibition (Fig. 6f ). Inhibition of autophagy resulted in decrease of RNF157 immunoreactivity in visceral adipocytes of both strains (Fig. 6b-e).

Up-regulation of cleaved caspase-3 signal pathway after inhibition of autophagic activity
Since autophagy promotes survival or once exhausted could activate apoptosis [26], cleaved caspase-3, an apoptosis marker, has been assessed in cultivated adipocytes in response to autophagy inhibition. Western blot analysis demonstrate significantly increased cleaved caspase-3 (17 kDa subunit) protein expression after autophagy inhibition in both adipocyte cell cultures of WOKW and LEW.1W rats predominantly in visceral adipocytes (Fig. 7a-c).

Discussion
Obesity and metabolic syndrome (MetS) have been linked with enhanced autophagic activity in AT [7]. Increased expression of autophagy genes correlates with visceral fat distribution, adipocyte hypertrophy, secretion of pro-inflammatory adipokine and contributes to adipose tissue (AT) inflammation [7,27,28]. There is a complex bidirectional relationship between autophagic activity and inflammation. Autophagy pathway induces or suppresses inflammatory responses, whereas inflammatory signals regulate the autophagic flux [12,29,30]. Inflammatory activation in obesity occurs principally in metabolic tissues including AT, liver and pancreas [5]. Expansion of AT with predominant adipocyte hypertrophy triggers the secretion of pro-inflammatory chemokines and causes obesity-induced inflammation. Among them, MCP-1, TNFα, interleukin IL-1 and IL-6   -d). a, b Western blot analysis revealed the decreased expression of adiponectin in visceral adipocytes of obese WOKW rats with MetS as compared to lean LEW.1W healthy rats. The adiponectin expression in subcutaneous adipocytes of both strains was slightly detected. The inhibition of PI3K-dependent autophagy pathway resulted in significantly upregulation of adiponectin levels, pronounced in adipocytes of WOKW rats. a, c, d Increased expression of MCP1 and TNFα protein was predominantly detected in visceral adipocytes of WOKW rats as compared to LEW.1W rats. These proteins could be significantly downregulated through autophagy inhibition (48 h) in both strains. Data from n = 6 are presented as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, according to the one-way analysis of variance together with the Newman-Keuls test. GAPDH was used as normalization control Fig. 5 Detection of adiponectin immunoreactivity after autophagy inhibition in visceral adipocytes of WOKW and LEW.1W rats. a-d″ Adionectin (red) with perilipin A (green) were detected in visceral adipocytes (higher autophagic activity) of WOKW and LEW.1W rats using double immunofluorescence staining Note the adiponectin immunoreactivity was lower in visceral adipocytes of WOKW rats than in LEW.1W rats (a-a″, c-c″). In accordance with Western blot data, the inhibition of PI3K signaling pathway and autophagic activity resulted in strikingly increase of adiponectin immunoreactivity (positive adipocytes) of both strains, with prevalence to WOKW rats (b-b″, d-d″). Nuclei are counterstained with DAPI (blue) Kosacka et al. Diabetol Metab Syndr (2018) 10:13 Fig. 6 Expression of autophagy related RNF157 protein in visceral and subcutaneous fat depots and cell cultures of WOKW and LEW.1W rats. a RNF157 mRNA expression was increased in the visceral and subcutaneous fat depots of obese WOKW rats with MetS as compared to lean LEW.1W healthy control rats. The higher mRNA expression of RNF157 was noted in visceral AT vs. subcutaneous in both strains. Data from four individuals per group are presented as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, according to the one-way analysis of variance together with the Newman-Keuls test. b-e Detection of RNF157 protein expression with immunofluorescence staining (green) in visceral adipocyte cell cultures of WOKW and LEW.1W. The higher immunoreactivity of RNF157 was found in adipocyte cultures of WOKW rats vs. LEW.1W rats. The markedly decrease of RNF157 expression was noted after autophagy inhibition with LY294002 (48 h). Nuclei are counterstained with DAPI (blue). f Representative Western blots and corresponding densitometrical analyses of RNF157. Western blot analysis revealed the significantly increased expression of RNF157 in visceral adipocytes of obese WOKW rats with MetS as compared to lean LEW.1W healthy rats. The RNF157 expression in subcutaneous adipocytes of both strains was slightly detected. The inhibition of PI3K-dependent autophagy pathway resulted in significantly downregulation of RNF157 levels, pronounced in adipocytes of WOKW rats. Data from n = 6 are presented as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, according to the one-way analysis of variance together with the Newman-Keuls test. GAPDH was used as normalization control Fig. 7 Regulation of the apoptosis marker cleaved caspase-3 under PI3K inhibition. Representative Western blots (a) and corresponding densitometrical analyses of cleaved caspase-3 (b-c). The cleaved caspase-3 expression in adipocyte cell cultures of both strains was slightly detected by Western blot. The inhibition with LY294002 (48 h) caused significantly increase of cleaved caspase-3 expression (17 kDa, mature form of enzyme) in visceral and subcutaneous adipocytes. An extract of Jurkat cells with cytochrome c-induced apoptosis was used in Western blot as the positive control for the occurrence of cleaved caspase-3 (a). Data from n = 6 are presented as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, according to the one-way analysis of variance together with the Newman-Keuls test. GAPDH was used as normalization control have been reported to promote obesity related-insulin resistance [28].
Obesity-related AT inflammation and insulin resistance result in a decrease of anti-inflammatory adipokines, such as adiponectin [28,31,32]. Slutsky, Rudich et al. have reported the association between decreased adiponectin and elevated autophagy in omental AT of patients with obesity [33]. However, obesity-related inflammation with up-regulated autophagy has been assumed as a destructive factor, the impact of autophagy as a protective mechanism in obesity is carefully discussed. Codogno and Meijer argued that initially obesity-induced insulin resistance functions as an adaptive mechanism to increase autophagy in order to protect cells against death [34]. Activated autophagy characterized by an increased expression of autophagy genes ATG5, LC3A, and LC3B as well as elevated autophagic flux in omental and subcutaneous adipose tissue has been reported in non-diabetic obese individuals more pronounced as in type 2 diabetes (T2D) patients [7,9,35]. Our previous data confirmed that autophagy contributes to AT inflammation in obesity and its related metabolic comorbidities [7,9,15,16]. Increased autophagy in obese and T2D patients compared to lean individuals is reflected by a higher infiltration of Iba-1 positive macrophages and up-regulation of TNFα and IL-6 expression in visceral AT. Interestingly, increased autophagy and inflammation in visceral AT of obese patients without T2D corresponded with inactivation of cleaved-caspase 3 dependent apoptosis pathway [9]. Autophagy occurring parallel to inflammation is assumed as a protective factor, since it does not invoke apoptosis. Correspondingly, we found a significant increased autophagy with macrophage infiltration in the sciatic nerves and visceral AT of adult, obese WOKW rats with MetS [15,16]. Notably, WOKW rats do not develop any age-related MetS complications such as diabetes or neuropathy. Therefore, we proposed that up-regulated autophagy exerts a protective role against development of diabetes and peripheral neurodegeneration in the WOKW rat model.
The present study highlights the impact of autophagy on adipocyte function in cultured adipocytes of obese WOKW rats with MetS. We demonstrate that inhibition of autophagy leads to enhanced expression of protective and inflammatory factors and induces the expression of cleaved caspase-3 apoptotic marker in adipocytes of WOKW rats as compared to the lean, healthy control LEW.1W rats.
In accordance with our previous findings, significantly up-regulated autophagy has been detected in cultured adipocytes of obese WOKW rats as compared to LEW.1W. We found a markedly higher expression of autophagic molecular markers, including Atg5-Atg12 complex, lipidated/cleaved form of LC3 (LC3-II), and down-regulation of mTOR protein, respectively.
Up-regulated autophagy in visceral adipocytes of WOKW rats vs. LEW.1W corresponds with an increase of RNF157 protein expression. RNF157 could be coregulated by autophagy mechanisms in adipose tissue or induce autophagy. Significantly higher mRNA and protein levels of RNF157 (about threefold) correlate with visceral AT mass, obesity and MetS parameters (WOKW vs. LEW.1W). Importantly, an inhibition of autophagic activity reduced the expression of RNF157 protein in AT of both strains. RNF157 is predominantly expressed in the brain, whereas non-neuronal tissues show little expression. This protein belongs to E3 ubiquitin RING ligases and is implicated in regulation of neuronal survival and morphology in cultured neurons [25]. In addition, a downstream component and substrate of RNF157the adaptor protein Fe65 was found to trigger neuronal apoptosis. These unexpected observations of AT specific RNF157 expression and its correlation with activated autophagy require further mechanistic studies. Among adipokines, adiponectin is an important modulator of glucose metabolism involved in insulin sensitivity and secretion [27]. Moreover, based on its anti-inflammatory properties, adiponectin has been classified as a protective adipokine [36]. Recently, up-regulated autophagy has been show to be implicated in impaired adiponectin secretion in omental AT of patients with obesity [33]. We have previously reported a tenfold reduced adiponectin mRNA in AT of obese WOKW rats as compared to healthy controls [19]. The present study revealed that autophagy significantly contributes to the regulation of adiponectin secretion in visceral and subcutaneous AT. Notably, lower adiponectin levels with up-regulated autophagy in cultured adipocytes (WOKW vs. LEW.1W) have been increased about fourfold after autophagy inhibition in adipocytes of WOKW rats. Whether the upregulation of autophagy genes with attenuated expression of adiponectin in AT of WOKW rats mediates anti-metabolic or protective actions is questionable. The WOKW rats develop complete metabolic syndrome, but are resistant to MetS complications.
In order to investigate autophagy dependent regulation of pro-inflammatory cytokines, we focused particularly on MCP-1 and TNF-α mRNA and protein expression in AT and cultivated adipocytes of WOKW rats with MetS and LEW.1W control healthy rats. MCP-1, a member of the chemokine family, is a crucial factor implicated in chronic inflammation of visceral AT. An increased level of MCP-1 in obesity links to accumulation of M1-proinflammatory macrophages in visceral AT [37]. MCP-1 plays a crucial role in the recruitment of monocytes and T lymphocytes into tissues [38]. The resident stromal cells, monocytes, endothelial cells, preadipocytes and adipocytes have been determined to MCP-1 expression. More recently, adipocyte progenitor cells have been identified as the initial cellular source of MCP-1 [37]. A potentially direct action of MCP-1 on autophagy is unknown. In the present study, we confirmed that MCP-1 expression in visceral adipocytes is directly regulated through autophagic activity. The MCP-1 mRNA levels were significantly higher in visceral and subcutaneous AT of WOKW rats as compared with LEW.1W rats. Correspondingly, higher MCP-1 protein expression was detected in cultured visceral adipocytes of WOKW rats vs. LEW.1W rats. The inhibition of autophagy resulted in significantly decrease of MCP-1 expression in visceral adipocytes of both strains. Differences between MCP-1 expression in AT (mRNA level) and in cultivated adipocytes (protein level) in WOKW rats vs. LEW.1W rats can be explained by the rare occurrence of macrophages or other components of stromal-vascular fraction in cell cultures as a source of MCP-1.
MCP-1, in concert with other pro-inflammatory cytokines such as TNF-α or IL-6 overexpressed by adipocytes in obesity and MetS, leads to insulin sensitivity. In the visceral adipocytes of WOKW rats, up-regulated autophagy corresponds to higher levels of TNFα, whereas the inhibition of autophagic activity significantly decreased TNFα expression. Perhaps, the autophagic process plays a regulatory function of cytokine dependent insulin sensitivity in visceral AT. The activation of autophagy pathways during inflammation has been reported to regulate pathogen clearance, antigen presentation, lymphocyte development and the production of proinflammatory cytokines [39]. The mechanistic relationship between autophagy and pro-inflammatory cytokine activation is dependent on the priority of occurring process, since one of these could precede the second.
In this study we demonstrate that an inhibition of autophagic activity significantly changed the expression of protective but also inflammatory factors and parallel markedly up-regulated the expression of cleaved caspase-3 in cultured adipocytes. However, the occurred changes were more evident in visceral adipocytes of obese WOKW rats than in LEW.1W healthy control rats. These results are compatible with our previous observation that increased autophagic activity protect from caspase-3 mediated apoptosis in AT of obese human and rats [9,15]. The biological consequences of manipulation of autophagy process are dependent on cell type and metabolic context and can exert opposing effects. It has been assumed that "basal autophagy" as an adipocyte survival mechanism is essential for completion of adipogenesis. Defects in autophagy or its inhibition cause the higher level of apoptosis in AT and cultivated adipocytes (our observation). Notably, the autophagy related protein (Atg)-5 knockout mice die within the first day after birth. Atg-7 knockout mice are insulin-sensitive and resistant to the development of obesity [40]. Contrary, up-regulated autophagy is implicated in the obesity state and obesityrelated inflammation. Therefore, autophagy represents distinctly regulated mechanism, which may protect against or develop obesity-associated AT dysfunction.

Conclusion
Based on our previously observations about up-regulated autophagy in visceral AT of obese patients and WOKW rats with MetS in correlation with inflammation but not with apoptosis and T2D, we postulated that autophagy as a key protective rather as destructive factor. Here, we show the consequences of preventing autophagic activity in adipocytes, predominantly in visceral AT of WOKW rats as markedly changes of pro-inflammatory and protective adipokines parallel with activated apoptosis. This fact highlights the autophagy process for future research of diabetes prevention in obesity.