GLUT3 promotes macrophage signaling and function via RAS-mediated endocytosis in atopic dermatitis and wound healing.

The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow-derived macrophages. Alternatively activated macrophages from the skin of patients with atopic dermatitis showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued in LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and costaining, and GLUT3 expression was significantly decreased in nonhealing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independently of its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL-4Rα subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain, and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.

Pre-implantation development of cattle embryos produced from fresh bull semen enriched for X- chromosome-bearing spermatozoa using a monoclonal antibody.

Immunological approaches are gaining attention as a convenient and economical method for sex-sorting mammalian spermatozoa. A monoclonal antibody (WholeMom™) has previously been reported to cause agglutination of Y-chromosome-bearing spermatozoa in frozen-thawed semen for gender preselection. However, its usefulness for gender preselection in fresh semen and subsequent in vitro fertilization (IVF) after freeze-thawing has not been reported. This study investigated the in vitro development of cattle embryos produced from fresh bull semen pre-treated with WholeMom™ monoclonal antibody. Results showed that antibody-treated, non-agglutinated spermatozoa (presumably X-chromosome-bearing spermatozoa) could fertilize cattle oocytes in vitro. However, embryos generated from non-agglutinated (enriched in X-chromosome-bearing spermatozoa) had a lower (p < 0.05) ability to cleave (66.4 ± 2.5% vs. 75.1 ± 3.3%) than those of non-treated control sperm. Nevertheless, the percentage of blastocysts developed from cleaved embryos did not differ (p > 0.05) between the groups (34.8 ± 3.7% vs. 35.8 ± 3.4%). Duplex PCR of blastocysts, using a bovine-specific universal primer pair and a Y-chromosome-specific primer pair, showed a sex ratio of 95.8% females from sex-sorted spermatozoa, which was higher than those of non-treated control spermatozoa (46.4%). In conclusion, the results of the present study suggest that monoclonal antibody-based enrichment of X- chromosome-bearing spermatozoa can be applied to fresh bull semen without compromising their post-fertilization early embryonic development to the blastocyst stage. Future studies should investigate the term development and sex ratio of calves from antibody-treated spermatozoa.

Red blood cells as glucose carriers to the human brain: Modulation of cerebral activity by erythrocyte exchange transfusion in Glut1 deficiency (G1D).

Red blood cells circulating through the brain are briefly but closely apposed to the capillary endothelium. We hypothesized that this contact provides a nearly direct pathway for metabolic substrate transfer to neural cells that complements the better characterized plasma to endothelium transfer. While brain function is considered independent of normal fluctuations in blood glucose concentration, this is not borne out by persons with glucose transporter I (GLUT1) deficiency (G1D). In them, encephalopathy is often ameliorated by meal or carbohydrate administration, and this enabled us to test our hypothesis: Since red blood cells contain glucose, and since the red cells of G1D individuals are also deficient in GLUT1, replacing them with normal donor cells via exchange transfusion could augment erythrocyte to neural cell glucose transport via mass action in the setting of unaltered erythrocyte count or plasma glucose abundance. This motivated us to perform red blood cell exchange in 3 G1D persons. There were rapid, favorable and unprecedented changes in cognitive, electroencephalographic and quality-of-life measures. The hypothesized transfer mechanism was further substantiated by in vitro measurement of direct erythrocyte to endothelial cell glucose flux. The results also indicate that the adult intellect is capable of significant enhancement without deliberate practice. ClinicalTrials.gov registration: NCT04137692 https://clinicaltrials.gov/ct2/show/NCT04137692.

Central role of Prominin-1 in lipid rafts during liver regeneration.

Prominin-1, a lipid raft protein, is required for maintaining cancer stem cell properties in hepatocarcinoma cell lines, but its physiological roles in the liver have not been well studied. Here, we investigate the role of Prominin-1 in lipid rafts during liver regeneration and show that expression of Prominin-1 increases after 2/3 partial hepatectomy or CCl4 injection. Hepatocyte proliferation and liver regeneration are attenuated in liver-specific Prominin-1 knockout mice compared to wild-type mice. Detailed mechanistic studies reveal that Prominin-1 interacts with the interleukin-6 signal transducer glycoprotein 130, confining it to lipid rafts so that STAT3 signaling by IL-6 is effectively activated. The overexpression of the glycosylphosphatidylinsositol-anchored first extracellular domain of Prominin-1, which is the domain that binds to GP130, rescued the proliferation of hepatocytes and liver regeneration in liver-specific Prominin-1 knockout mice. In summary, Prominin-1 is upregulated in hepatocytes during liver regeneration where it recruits GP130 into lipid rafts and activates the IL6-GP130-STAT3 axis, suggesting that Prominin-1 might be a promising target for therapeutic applications in liver transplantation.

Hepatocyte-specific Prominin-1 protects against liver injury-induced fibrosis by stabilizing SMAD7.

Prominin-1 (PROM1), also known as CD133, is expressed in hepatic progenitor cells (HPCs) and cholangiocytes of the fibrotic liver. In this study, we show that PROM1 is upregulated in the plasma membrane of fibrotic hepatocytes. Hepatocellular expression of PROM1 was also demonstrated in mice (Prom1CreER; R26TdTom) in which cells expressed TdTom under control of the Prom1 promoter. To understand the role of hepatocellular PROM1 in liver fibrosis, global and liver-specific Prom1-deficient mice were analyzed after bile duct ligation (BDL). BDL-induced liver fibrosis was aggravated with increased phosphorylation of SMAD2/3 and decreased levels of SMAD7 by global or liver-specific Prom1 deficiency but not by cholangiocyte-specific Prom1 deficiency. Indeed, PROM1 prevented SMURF2-induced SMAD7 ubiquitination and degradation by interfering with the molecular association of SMAD7 with SMURF2. We also demonstrated that hepatocyte-specific overexpression of SMAD7 ameliorated BDL-induced liver fibrosis in liver-specific Prom1-deficient mice. Thus, we conclude that PROM1 is necessary for the negative regulation of TGFß signaling during liver fibrosis.

Association between anemia and outcome in patients hospitalized for acute heart failure syndromes: findings from Beijing Acute Heart Failure Registry (Beijing AHF Registry).

Whether the anemia increases the risk of mortality in patients with acute heart failure (AHF) remains unclear. This study aims to explore the relationship between anemia and outcomes in patients with AHF including subgroup analysis. This study included 3279 patients with hemoglobin available from the Beijing Acute Heart Failure Registry (Beijing AHF Registry) study. The primary endpoint was all-cause mortality in 1 year, and the secondary endpoint was 1-year all-cause events including all-cause death and readmission. Logistic regression models were applied to describe related variables of anemia in patients with AHF. Multivariate Cox proportional hazards models described associations of anemia with clinical outcomes in the overall cohort and subgroups. 45.4% of the patients were found anemic. They were older and had more comorbidities than non-anemic patients. Variables including older age, female, chronic kidney dysfunction (CKD), lower hematocrit, lower albumin, with loop diuretics applied, without beta-blockers, angiotensin-converting enzyme inhibitors /angiotensin receptor blockers (ACEIs/ARBs) and spironolactone applied in the emergency department (ED) were associated with anemia in AHF patients. Anemic patients had higher 1-year mortality (38.4% vs. 27.2%, p < 0.0001) and 1-year events rates (63.2% vs. 56.7%, p < 0.0001). After adjusted for covariates, anemia was associated with the increase of 1-year mortality (hazard ratio [HR] 1.278; 95% confidence interval [CI] 1.114-1.465; p = 0.0005) and 1-year events (HR 1.136; 95% CI 1.025-1.259; p = 0.0154). The severer anemia patients had higher risks both of 1-year mortality and events. In the subgroup analysis, the independent associations of anemia with 1-year mortality were shown in the subgroups including age < 75 years, male, body mass index < 25 kg/m2 and BMI ≥ 25 kg/m2, New York Heart Association (NYHA) functional class I-II and NYHA functional class III-IV, with and without cardiovascular ischemia, heart rate (HR) < 100 bpm and HR ≥ 100 bpm, systolic blood pressure (SBP) < 120 mmHg and SBP ≥ 120 mmHg, left ventricular ejection fraction (LVEF) < 40% and LVEF ≥ 40%, serum creatinine (Scr) < 133 umol/l, and with diuretics use, with and without beta-blockers use, without ACEIs/ARBs use in the ED. Anemia is associated with older age, female, CKD, volume overload, malnutrition, with loop diuretics, without beta-blockers, ACEIs/ARBs and spironolactone administration, and higher mortality and readmission in AHF. The risk associations are particular significantly obvious in younger, male, overweight, preserved LVEF, lower Scr, with diuretics and beta-blockers, without ACEIs/ARBs administration subgroups.Clinical trial No. ChiCTR-RIC-17014222.

Prominin-1-Radixin axis controls hepatic gluconeogenesis by regulating PKA activity.

Prominin-1 (Prom1) is a major cell surface marker of cancer stem cells, but its physiological functions in the liver have not been elucidated. We analyzed the levels of mRNA transcripts in serum-starved primary WT (Prom1+/+ ) and KO (Prom1-/- ) mouse hepatocytes using RNA sequencing (RNA-seq) data, and found that CREB target genes were downregulated. This initial observation led us to determine that Prom1 deficiency inhibited cAMP response element-binding protein (CREB) activation and gluconeogenesis, but not cyclic AMP (cAMP) accumulation, in glucagon-, epinephrine-, or forskolin-treated liver tissues and primary hepatocytes, and mitigated glucagon-induced hyperglycemia. Because Prom1 interacted with radixin, Prom1 deficiency prevented radixin from localizing to the plasma membrane. Moreover, systemic adenoviral knockdown of radixin inhibited CREB activation and gluconeogenesis in glucagon-treated liver tissues and primary hepatocytes, and mitigated glucagon-elicited hyperglycemia. Based on these results, we conclude that Prom1 regulates hepatic PKA signaling via radixin functioning as an A kinase-anchored protein (AKAP).

Integrin α6ß4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues.

Cellular senescence refers to an irreversible growth arrest that is triggered by various intrinsic and extrinsic stresses. Many recent studies have demonstrated that cellular senescence plays a crucial role in the regression of tumors exposed to ionizing radiation (IR), but the underlying mechanism remains unknown. Here we show that the activation of integrin ß4 is essential for IR-induced cellular senescence. IR treatment results in the phosphorylation of integrin ß4 at tyrosine residue 1510, leading to activation of the integrin α6ß4-Src-AKT signaling pathway. We further reveal that the IR-induced phosphorylation of integrin ß4 is regulated by the cholesterol content and membrane fluidity. We also find that IR-induced p53-caspase signaling is independent of integrin α6ß4-Src-AKT signaling. Finally, we show that siRNA- or inhibitor-mediated blockade of integrin α6ß4-Src-AKT signaling switches the post-irradiation fate from senescence to apoptosis, under p53 activated condition, in both cancer cells and tumor tissues of xenograft mice. On the basis of our finding that, integrin α6ß4 is specifically activated and acts primarily to induce premature senescence in irradiated cancer cells, we propose that this integrin may be a valuable target and biomarker for radiotherapy.

Characteristics, Management, and Outcomes of Acute Heart Failure in the Emergency Department: A Multicenter Registry Study with 1-year Follow-up in a Chinese Cohort in Beijing.

BACKGROUND: The emergency department (ED) has a pivotal influence on the management of acute heart failure (AHF), but data concerning current ED management are scarce. This Beijing AHF Registry Study investigated the characteristics, ED management, and short- and long-term clinical outcomes of AHF. METHODS: This prospective, multicenter, observational study consecutively enrolled 3335 AHF patients who visited 14 EDs in Beijing from January 1, 2011, to September 23, 2012. Baseline data on characteristics and management were collected in the EDs. Follow-up data on death and readmissions were collected until November 31, 2013, with a response rate of 92.80%. The data were reported as median (interquartile range) for the continuous variables, or as number (percentage) for the categorical variables. RESULTS: The median age of the enrolled patients was 71 (58-79) years, and 46.84% were women. In patients with AHF, coronary heart disease (43.27%) was the most common etiology, and myocardium ischemia (30.22%) was the main precipitant. Most of the patients in the ED received intravenous treatments, including diuretics (79.28%) and vasodilators (74.90%). Fewer patients in the ED received neurohormonal antagonists, and 25.94%, 31.12%, and 33.73% of patients received angiotensin converting enzyme inhibitors/angiotensin receptor blockers, beta-blockers, and spironolactone, respectively. The proportions of patients who were admitted, discharged, left against medical advice, and died were 55.53%, 33.58%, 7.08%, and 3.81%, respectively. All-cause mortalities at 30 days and 1 year were 15.30% and 32.27%, respectively. CONCLUSIONS: Substantial details on characteristics and ED management of AHF were investigated. The clinical outcomes of AHF patients were dismal. Thus, further investigations of ED-based therapeutic approaches for AHF are needed.

Caveolin-1 deficiency induces premature senescence with mitochondrial dysfunction.

Paradoxical observations have been made regarding the role of caveolin-1 (Cav-1) during cellular senescence. For example, caveolin-1 deficiency prevents reactive oxygen species-induced cellular senescence despite mitochondrial dysfunction, which leads to senescence. To resolve this paradox, we re-addressed the role of caveolin-1 in cellular senescence in human diploid fibroblasts, A549, HCT116, and Cav-1-/- mouse embryonic fibroblasts. Cav-1 deficiency (knockout or knockdown) induced cellular senescence via a p53-p21-dependent pathway, downregulating the expression level of the cardiolipin biosynthesis enzymes and then reducing the content of cardiolipin, a critical lipid for mitochondrial respiration. Our results showed that Cav-1 deficiency decreased mitochondrial respiration, reduced the activity of oxidative phosphorylation complex I (CI), inactivated SIRT1, and decreased the NAD+ /NADH ratio. From these results, we concluded that Cav-1 deficiency induces premature senescence via mitochondrial dysfunction and silent information regulator 2 homologue 1 (SIRT1) inactivation.

Mitochondrial complex I deficiency enhances skeletal myogenesis but impairs insulin signaling through SIRT1 inactivation.

To address whether mitochondrial biogenesis is essential for skeletal myogenesis, C2C12 myogenesis was investigated after knockdown of NADH dehydrogenase (ubiquintone) flavoprotein 1 (NDUFV1), which is an oxidative phosphorylation complex I subunit that is the first subunit to accept electrons from NADH. The NDUFVI knockdown enhanced C2C12 myogenesis by decreasing the NAD(+)/NADH ratio and subsequently inactivating SIRT1 and SIRT1 activators (pyruvate, SRT1720, and resveratrol) abolished the NDUFV1 knockdown-induced myogenesis enhancement. However, the insulin-elicited activation of insulin receptor ß (IRß) and insulin receptor substrate-1 (IRS-1) was reduced with elevated levels of protein-tyrosine phosphatase 1B after NDUFV1 knockdown in C2C12 myotubes. The NDUFV1 knockdown-induced blockage of insulin signaling was released by protein-tyrosine phosphatase 1B knockdown in C2C12 myotubes, and we found that NDUFV1 or SIRT1 knockdown did not affect mitochondria biogenesis during C2C12 myogenesis. Based on these data, we can conclude that complex I dysfunction-induced SIRT1 inactivation leads to myogenesis enhancement but blocks insulin signaling without affecting mitochondria biogenesis.