Celastrol directly binds with VAMP7 and RAB7 to inhibit autophagy and induce apoptosis in preadipocytes.

Obesity is one of the most prevalent chronic metabolic diseases, and induction of apoptosis in preadipocytes and adipocytes is a potential strategy to treat obesity. Celastrol represents one of the most robust anti-obesity phytochemicals so far, yet its direct binding target remains elusive. Here, we determined that celastrol could induce apoptosis in preadipocytes via mitochondrial mediated pathway. Further study clarified that celastrol inhibited the fusion of autophagosome and lysosome to prohibit autophagy, leading to cell apoptosis. By conducting virtual screening and genetic manipulation, we verified that overexpression of VAMP7 and RAB7 could block the effects of celastrol on inhibiting autophagy and inducing apoptosis. The Surface Plasmon Resonance study confirmed the direct binding of celastrol with VAMP7 and RAB7. The functional study illustrated the inhibition of RAB7 GTPase activity after celastrol treatment. Moreover, celastrol induced comparable apoptosis in murine epididymal adipose tissue, human preadipocytes and adipocytes, but not in human hepatocytes. An inhibitory effect on differentiation of human primary visceral preadipocytes was also observed. In conclusion, celastrol exhibited inhibitory effect of autophagy via direct binding with VAMP7 and RAB7, leading to an increase in preadipocytes apoptosis. These results advance our understanding in the potential application of celastrol in treating obesity.

Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry.

Coronavirus disease 2019 (COVID-19) represents a systemic disease that may cause severe metabolic complications in multiple tissues including liver, kidney, and cardiovascular system. However, the underlying mechanisms and optimal treatment remain elusive. Our study shows that impairment of ACE2 pathway is a key factor linking virus infection to its secondary metabolic sequelae. By using structure-based high-throughput virtual screening and connectivity map database, followed with experimental validations, we identify imatinib, methazolamide, and harpagoside as direct enzymatic activators of ACE2. Imatinib and methazolamide remarkably improve metabolic perturbations in vivo in an ACE2-dependent manner under the insulin-resistant state and SARS-CoV-2-infected state. Moreover, viral entry is directly inhibited by these three compounds due to allosteric inhibition of ACE2 binding to spike protein on SARS-CoV-2. Taken together, our study shows that enzymatic activation of ACE2 via imatinib, methazolamide, or harpagoside may be a conceptually new strategy to treat metabolic sequelae of COVID-19.

COVID-19 induces new-onset insulin resistance and lipid metabolic dysregulation via regulation of secreted metabolic factors.

Abnormal glucose and lipid metabolism in COVID-19 patients were recently reported with unclear mechanism. In this study, we retrospectively investigated a cohort of COVID-19 patients without pre-existing metabolic-related diseases, and found new-onset insulin resistance, hyperglycemia, and decreased HDL-C in these patients. Mechanistically, SARS-CoV-2 infection increased the expression of RE1-silencing transcription factor (REST), which modulated the expression of secreted metabolic factors including myeloperoxidase, apelin, and myostatin at the transcriptional level, resulting in the perturbation of glucose and lipid metabolism. Furthermore, several lipids, including (±)5-HETE, (±)12-HETE, propionic acid, and isobutyric acid were identified as the potential biomarkers of COVID-19-induced metabolic dysregulation, especially in insulin resistance. Taken together, our study revealed insulin resistance as the direct cause of hyperglycemia upon COVID-19, and further illustrated the underlying mechanisms, providing potential therapeutic targets for COVID-19-induced metabolic complications.

Resveratrol Promotes Angiogenesis in a FoxO1-Dependent Manner in Hind Limb Ischemia in Mice.

Critical limb ischemia (CLI) is a severe form of peripheral artery diseases (PAD) and seriously endangers the health of people. Therapeutic angiogenesis represents an important treatment strategy for CLI; various methods have been applied to enhance collateral circulation. However, the current development drug therapy to promote angiogenesis is limited. Resveratrol (RSV), a polyphenol compound extracted from plants, has various properties such as anti-oxidative, anti-inflammatory and anti-cancer effects. Whether RSV exerts protective effects on CLI remains elusive. In the current study, we demonstrated that oral intake of RSV significantly improved hind limb ischemia in mice, and increased the expression of phosphorylated Forkhead box class-O1 (FoxO1). RSV treatment in human umbilical vein endothelial cells (HUVECs) could increase the phosphorylation of FoxO1 and its cytoplasmic re-localization to promote angiogenesis. Then we manipulated FoxO1 in HUVECs to further verify that the effect of RSV on angiogenesis is in a FoxO1-dependent manner. Furthermore, we performed metabolomics to screen the metabolic pathways altered upon RSV intervention. We found that the pathways of pyrimidine metabolism, purine metabolism, as well as alanine, aspartate and glutamate metabolism, were highly correlated with the beneficial effects of RSV on the ischemic muscle. This study provides a novel direction for the medical therapy to CLI.

Glycoursodeoxycholic Acid Ameliorates Atherosclerosis and Alters Gut Microbiota in Apolipoprotein E-Deficient Mice.

Background Although glycoursodeoxycholic acid (GUDCA) has been associated with the improvement of metabolic disorders, its effect on atherosclerosis remains elusive. This study aimed to investigate the role of GUDCA in the development of atherosclerosis and its potential mechanisms. Methods and Results Human THP-1 macrophages were used to investigate the effect of GUDCA on oxidized low-density lipoprotein-induced foam cell formation in vitro. We found that GUDCA downregulated scavenger receptor A1 mRNA expression, reduced oxidized low-density lipoprotein uptake, and inhibited macrophage foam cell formation. In an in vivo study, apolipoprotein E-deficient mice were fed a Western diet for 10 weeks to induce atherosclerosis, and then were gavaged once daily with or without GUDCA for 18 weeks. Parameters of systemic metabolism and atherosclerosis were detected. We found that GUDCA improved cholesterol homeostasis and protected against atherosclerosis progression as evidenced by reduced plaque area along with lipid deposition, ameliorated local chronic inflammation, and elevated plaque stability. In addition, 16S rDNA sequencing showed that GUDCA administration partially normalized the Western diet-associated gut microbiota dysbiosis. Interestingly, the changes of bacterial genera (Alloprevotella, Parabacteroides, Turicibacter, and Alistipes) modulated by GUDCA were correlated with the plaque area in mice aortas. Conclusions Our study for the first time indicates that GUDCA attenuates the development of atherosclerosis, probably attributable to the inhibition of foam cell formation, maintenance of cholesterol homeostasis, and modulation of gut microbiota.

Meta-analysis of redo stenting versus endarterectomy for in-stent stenosis after carotid artery stenting.

OBJECTIVE: The development of in-stent restenosis (ISR) hinders the long-term patency of carotid artery stenting (CAS), yet no optimal treatment has been established. In the present study, we compared the outcomes of redo CAS (rCAS) and carotid endarterectomy (CEA) for ISR. METHODS: A systematic search using the terms "in-stent restenosis," "carotid endarterectomy," and "carotid artery stenting" was conducted in the PubMed, Embase, and Cochrane databases. Studies reporting perioperative stroke, death, and other important complications of rCAS or CEA for ISR after previous CAS with four or more patients were included. Pooled and sensitivity analyses were conducted to synthesize and compare estimates of the outcomes. RESULTS: A total of 11 studies with 1057 patients who had undergone rCAS (n = 894) or CEA (n = 163) met the inclusion criteria. The CEA group had a significantly greater proportion of symptomatic patients (rCAS vs CEA, 30.4% vs 42.1%; P < .01). The duration from primary CAS to reintervention was relatively longer in the CEA group (rCAS vs CEA, median, 8.8 months [range, 3-26 months] vs 19.9 months [range, 0-54 months]). In the rCAS group, a greater proportion of patients had hypertension, hypercholesterolemia, and coronary artery disease and had received antiplatelet therapy before reintervention. Because of insufficient data or a low incidence, the only complications feasible for further analysis were restenosis, myocardial infarction, cranial nerve injury, and neck hematoma. No significant differences were found in the primary end point of mortality/stroke event-free rate (rCAS vs CEA, 99% vs 98%; P > .05) or other secondary end points (event-free restenosis, 100% vs 100%; event-free myocardial infarction, 100% vs 98%; event-free cranial nerve injury, 100% vs 98%; event-free neck hematoma, 100% vs 100% for rCAS vs CEA; P > .05 for all). CONCLUSIONS: rCAS is commonly used to treat patients with severe and/or symptomatic ISR after primary CAS. Although the endovascular approach is less invasive, both rCAS and CEA can be performed safely with similar short- and midterm outcomes of stroke, death, and surgery-related complications.

Enhanced autophagy alleviates injury during hindlimb ischemia/reperfusion in mice.

Previous studies examining whether autophagy has a protective or deleterious role during ischemia/reperfusion (I/R) injury have reported a varying role in different organs and remains a matter of debate. The aim of the current study was to explore the role of autophagy in hindlimb I/R injury in a murine model. An increase in apoptosis was observed in vitro, in C2C12 myoblast cells, following hypoxia/reoxygenation (H/R), while downregulation of autophagic flux was induced by chloroquine as compared with the vehicle group under hypoxia and H/R conditions. In vivo, an increase in severe damage of gastrocnemius muscles was observed in the I/R and ischemia groups compared with the control group, was more severe in the I/R group compared with the ischemia group. Electron microscopy revealed an increased number of autophagosomes in the ischemia group, whereas a reduced number was detected in the I/R group. Following administration of rapamycin, the infarct size ratio and cell apoptosis was significantly reduced, while the amount of autophagosomes significantly increased in the ischemic phase. In conclusion, autophagy is upregulated in the ischemia phase and downregulated in the reperfusion phase. Notably, upregulation of autophagy via rapamycin intervention during ischemia alleviated skeletal muscle damage, suggesting a potential protective role during hindlimb I/R injury.

The oncogene c-Jun impedes somatic cell reprogramming.

Oncogenic transcription factors are known to mediate the conversion of somatic cells to tumour or induced pluripotent stem cells (iPSCs). Here we report c-Jun as a barrier for iPSC formation. c-Jun is expressed by and required for the proliferation of mouse embryonic fibroblasts (MEFs), but not mouse embryonic stem cells (mESCs). Consistently, c-Jun is induced during mESC differentiation, drives mESCs towards the endoderm lineage and completely blocks the generation of iPSCs from MEFs. Mechanistically, c-Jun activates mesenchymal-related genes, broadly suppresses the pluripotent ones, and derails the obligatory mesenchymal to epithelial transition during reprogramming. Furthermore, inhibition of c-Jun by shRNA, dominant-negative c-Jun or Jdp2 enhances reprogramming and replaces Oct4 among the Yamanaka factors. Finally, Jdp2 anchors 5 non-Yamanaka factors (Id1, Jhdm1b, Lrh1, Sall4 and Glis1) to reprogram MEFs into iPSCs. Our studies reveal c-Jun as a guardian of somatic cell fate and its suppression opens the gate to pluripotency.

An antiapoptotic role of sorting nexin 7 is required for liver development in zebrafish.

UNLABELLED: Sorting nexin (SNX) family proteins are best characterized for their abilities to regulate protein trafficking during processes such as endocytosis of membrane receptors, endosomal sorting, and protein degradation, but their in vivo functions remain largely unknown. We started to investigate the biological functions of SNXs using the zebrafish model. In this study, we demonstrated that SNX7 was essential for embryonic liver development. Hepatoblasts were specified normally, and the proliferation of these cells was not affected when SNX7 was knocked down by gene-specific morpholinos; however, they underwent massive apoptosis during the early budding stage. SNX7 mainly regulated the survival of cells in the embryonic liver and did not affect the viability of cells in other endoderm-derived organs. We further demonstrated that down-regulation of SNX7 by short interfering RNAs induced apoptosis in cell culture. At the molecular level, the cellular FLICE-like inhibitory protein (c-FLIP)/caspase 8 pathway was activated when SNX7 was down-regulated. Furthermore, overexpression of c-FLIP(S) was able to rescue the SNX7 knockdown-induced liver defect. CONCLUSION: SNX7 is a liver-enriched antiapoptotic protein that is indispensable for the survival of hepatoblasts during zebrafish early embryogenesis.

Towards an optimized culture medium for the generation of mouse induced pluripotent stem cells.

Generation of induced pluripotent stem cells from somatic cells using defined factors has potential relevant applications in regenerative medicine and biology. However, this promising technology remains inefficient and time consuming. We have devised a serum free culture medium termed iSF1 that facilitates the generation of mouse induced pluripotent stem cells. This optimization of the culture medium is sensitive to the presence of Myc in the reprogramming factors. Moreover, we could reprogram meningeal cells using only two factors Oct4/Klf4. Therefore, iSF1 represents a basal medium that may be used for mechanistic studies and testing new reprogramming approaches.

Porcine induced pluripotent stem cells may bridge the gap between mouse and human iPS.

Recently, three independent laboratories reported the generation of induced pluripotent stem cells (iPSCs) from pig (Sus scrofa). This finding sums to the growing list of species (mouse, human, monkey, and rat, in this order) for which successful reprogramming using exogenous factors has been achieved, and multiple others are possibly forthcoming. But apart from demonstrating the universality of the network identified by Shinya Yamanaka, what makes the porcine model so special? On one side, pigs are an agricultural commodity and have an easy and affordable maintenance compared with nonhuman primates that normally need to be imported. On the other side, resemblance (for example, size of organs) of porcine and human physiology is striking and because pigs are a regular source of food the ethical concerns that still remain in monkeys are not applicable. Besides, the prolonged lifespan of pigs compared with other domestic species can allow exhaustive follow up of side effects after transplantation. Porcine iPSCs may thus fill the gap between the mouse model, which due to its ease is preferred for mechanistic studies, and the first clinical trials using iPSCs in humans. However, although these studies are relevant and have created significant interest they face analogous problems that we discuss herein together with potential new directions.

Generation of induced pluripotent stem cell lines from Tibetan miniature pig.

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model.