Atf4 protects islet ß-cell identity and function under acute glucose-induced stress but promotes ß-cell failure in the presence of free fatty acid.

Glucolipotoxicity, caused by combined hyperglycemia and hyperlipidemia, results in ß-cell failure and type 2 diabetes (T2D) via cellular stress-related mechanisms. Activating transcription factor 4 (Atf4) is an essential effector of stress response. We show here that Atf4 expression in ß-cells is dispensable for glucose homeostasis in young mice, but it is required for ß-cell function during aging and under obesity-related metabolic stress. Henceforth, aged Atf4- deficient ß-cells display compromised secretory function under acute hyperglycemia. In contrast, they are resistant to acute free fatty acid-induced loss-of identity and dysfunction. At molecular level, Atf4 -deficient ß-cells down-regulate genes involved in protein translation, reducing ß-cell identity gene products under high glucose. They also upregulate several genes involved in lipid metabolism or signaling, likely contributing to their resistance to free fatty acid-induced dysfunction. These results suggest that Atf4 activation is required for ß-cell identity and function under high glucose, but this paradoxically induces ß-cell failure in the presence of high levels of free fatty acids. Different branches of Atf4 activity could be manipulated for protecting ß-cells from metabolic stress-induced failure. Highlights: Atf4 is dispensable in ß-cells in young miceAtf4 protects ß-cells under high glucoseAtf4 exacerbate fatty acid-induced ß-cell defectsAtf4 activates translation but depresses lipid-metabolism.

Metabolic stress levels influence the ability of the myelin transcription factors to regulate ß-cell identity and survival.

A hallmark of type 2 diabetes (T2D) is endocrine islet ß-cell failure, which can occur via cell dysfunction, loss-of-identity, and/or death. How each is induced remains largely unknown. Here, we use mouse ß-cells that are deficient for Myelin transcription factors (Myt TFs, including Myt1, 2, and 3) to address this question. We have reported that inactivating all three Myt genes in pancreatic progenitor cells (MytPancΔ) causes ß-cell failure and late onset diabetes in mice. Their lower expression in human ß-cells is correlated with ß-cell dysfunction and SNPs in MYT2 and MYT3 are associated in higher risk of T2D. We now show that these Myt TF-deficient postnatal ß-cells also de-differentiate by reactivating several progenitor markers. Intriguingly, mosaic Myt TF inactivation in only a portion of islet ß-cells does not results in overt diabetes, but this creates a condition where Myt TF-deficient ß-cells stay alive while activating several markers of Ppy-expressing islet cells. By transplanting MytPancΔ islets into the anterior eye chambers of immune-compromised mice, we directly show that glycemic and obesity-related conditions influence cell fate, with euglycemia inducing several Ppy+ cell markers while hyperglycemia and insulin resistance inducing additional cell death. These findings suggest that the observed ß-cell defects in T2D depend on not only their inherent genetic/epigenetic defects, but also the metabolic load.

Qi Huang Fang improves intestinal barrier function and intestinal microbes in septic mice through NLRP3 inflammasome-mediated cellular pyroptosis.

OBJECTIVE: Sepsis has a high incidence, morbidity, and mortality rate and is a great threat to human safety. Gut health plays an important role in sepsis development. Qi Huang Fang (QHF) contains astragalus, rhubarb, zhishi, and atractylodes. It is used to treat syndromes of obstructive qi and deficiency of righteousness. This study aimed to investigate whether QHF improves intestinal barrier function and microorganisms in mice through NLRP3 inflammatory vesicle-mediated cellular focal death. METHODS: A mouse model of sepsis was constructed by cecal ligation and puncture (CLP) of specific pathogen-free (SPF)-grade C57BL/6 mice after continuous gavage of low, medium, and high doses of astragalus formula or probiotics for 4 weeks. Twenty-four hours postoperatively, the mechanism of action of QHF in alleviating septic intestinal dysfunction and restoring intestinal microecology, thereby alleviating intestinal injury, was evaluated by pathological observation, immunohistochemistry, western blotting, ELISA, and 16S rDNA high-throughput sequencing. RESULTS: Different doses of QHF and probiotics ameliorated intestinal injury and reduced colonic apoptosis in mice to varying degrees (P < 0.05). Meanwhile, different doses of QHF and probiotics were able to reduce the serum levels of IL-6, IL-1ß, and TNF-α (P < 0.05); down-regulate the protein expression of NLRP3, caspase-1, and caspase-11 (P < 0.05); and up-regulate the protein expression of zonula occluden-1 (ZO-1) and occludin (P < 0.05), which improved the intestinal barrier function in mice. In addition, QHF decreased the relative abundance of harmful bacteria (Firmicutes, Muribaculaceae, Campilobacterota, Helicobacter, and Alistipes) and increased the relative abundance of beneficial bacteria (Bacteroidetes and Actinobacteria) (P < 0.05). CONCLUSION: QHF improves intestinal barrier function and gut microbiology in mice via NLRP3 inflammasome-mediated cellular pyroptosis.

Endocrine islet ß-cell subtypes with differential function are derived from biochemically distinct embryonic endocrine islet progenitors that are regulated by maternal nutrients.

Endocrine islet b cells comprise heterogenous cell subsets. Yet when/how these subsets are produced and how stable they are remain unknown. Addressing these questions is important for preventing/curing diabetes, because lower numbers of b cells with better secretory function is a high risk of this disease. Using combinatorial cell lineage tracing, scRNA-seq, and DNA methylation analysis, we show here that embryonic islet progenitors with distinct gene expression and DNA methylation produce b-cell subtypes of different function and viability in adult mice. The subtype with better function is enriched for genes involved in vesicular production/trafficking, stress response, and Ca2+-secretion coupling, which further correspond to differential DNA methylation in putative enhancers of these genes. Maternal overnutrition, a major diabetes risk factor, reduces the proportion of endocrine progenitors of the b-cell subtype with better-function via deregulating DNA methyl transferase 3a. Intriguingly, the gene signature that defines mouse b-cell subtypes can reliably divide human cells into two sub-populations while the proportion of b cells with better-function is reduced in diabetic donors. The implication of these results is that modulating DNA methylation in islet progenitors using maternal food supplements can be explored to improve b-cell function in the prevention and therapy of diabetes.

Overall survival and short-term efficacy analysis of cervical squamous cell carcinoma with skeletal muscle and 18F-FDG PET/CT parameters.

2-[18F]fluoro-2-deoxy-d-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) can provide tumor biological metabolism and skeletal muscle composition information. The aim of this study was to evaluate overall survival (OS) and short-term efficacy of cervical squamous cell carcinoma combining tumor biological metabolism and skeletal muscle composition parameters. Eighty two patients with cervical squamous cell carcinoma were included in the study, who received 18F-FDG PET/CT scans before treatment. Clinical characteristics, tumor biological metabolism parameters [standardized uptake value, metabolic tumor volume (MTV), total lesion glycolysis, heterogeneity of tumors, etc.] and body composition parameters were recorded. The survival analysis of cervical squamous cell carcinoma patients was performed by univariate and multivariate analysis. A combined model included clinical indicators, tumor metabolism parameters and sarcopenia was constructed to evaluate OS of patients. According to the Response Evaluation Criteria in Solid Tumours version 1.1, the relationship between sarcopenia with tumor metabolism parameters and short-term efficacy was investigated in subgroup. The results indicate that sarcopenia and high value of the sum of MTV of lesions and metastases (MTVtotal) were poor prognostic factors in patients with cervical squamous cell carcinoma. The combination of sarcopenia, MTVtotal and clinical factors provided an improved prediction of OS especially in the long term after treatment. Nutritional status of the patients and tumor metabolism may not affect the short-term efficacy of chemoradiotherapy in cervical squamous cell carcinoma patients.

DUSP1 interacts with and dephosphorylates VCP to improve mitochondrial quality control against endotoxemia-induced myocardial dysfunction.

Dual specificity phosphatase 1 (DUSP1) and valosin-containing protein (VCP) have both been reported to regulate mitochondrial homeostasis. However, their impact on mitochondrial quality control (MQC) and myocardial function during LPS-induced endotoxemia remains unclear. We addressed this issue by modeling LPS-induced endotoxemia in DUSP1 transgenic (DUSP1TG) mice and in cultured DUSP1-overexpressing HL-1 cardiomyocytes. Accompanying characteristic structural and functional deficits, cardiac DUSP1 expression was significantly downregulated following endotoxemia induction in wild type mice. In contrast, markedly reduced myocardial inflammation, cardiomyocyte apoptosis, cardiac structural disorder, cardiac injury marker levels, and normalized systolic/diastolic function were observed in DUSP1TG mice. Furthermore, DUSP1 overexpression in HL-1 cells significantly attenuated LPS-mediated mitochondrial dysfunction by preserving MQC, as indicated by normalized mitochondrial dynamics, improved mitophagy, enhanced biogenesis, and attenuated mitochondrial unfolded protein response. Molecular assays showed that VCP was a substrate of DUSP1 and the interaction between DUSP1 and VCP primarily occurred on the mitochondria. Mechanistically, DUSP1 phosphatase domain promoted the physiological DUSP1/VCP interaction which prevented LPS-mediated VCP Ser784 phosphorylation. Accordingly, transfection with a phosphomimetic VCP mutant abolished the protective actions of DUSP1 on MQC and aggravated inflammation, apoptosis, and contractility/relaxation capacity in HL-1 cardiomyocytes. These findings support the involvement of the novel DUSP1/VCP/MQC pathway in the pathogenesis of endotoxemia-caused myocardial dysfunction.

Microtubules and Gαo-signaling modulate the preferential secretion of young insulin secretory granules in islet ß cells via independent pathways.

For sustainable function, each pancreatic islet ß cell maintains thousands of insulin secretory granules (SGs) at all times. Glucose stimulation induces the secretion of a small portion of these SGs and simultaneously boosts SG biosynthesis to sustain this stock. The failure of these processes, often induced by sustained high-insulin output, results in type 2 diabetes. Intriguingly, young insulin SGs are more likely secreted during glucose-stimulated insulin secretion (GSIS) for unknown reasons, while older SGs tend to lose releasability and be degraded. Here, we examine the roles of microtubule (MT) and Gαo-signaling in regulating the preferential secretion of young versus old SGs. We show that both MT-destabilization and Gαo inactivation results in more SGs localization near plasma membrane (PM) despite higher levels of GSIS and reduced SG biosynthesis. Intriguingly, MT-destabilization or Gαo-inactivation results in higher secretion probabilities of older SGs, while combining both having additive effects on boosting GSIS. Lastly, Gαo inactivation does not detectably destabilize the ß-cell MT network. These findings suggest that Gαo and MT can modulate the preferential release of younger insulin SGs via largely parallel pathways.

Myt Transcription Factors Prevent Stress-Response Gene Overactivation to Enable Postnatal Pancreatic ß Cell Proliferation, Function, and Survival.

Although cellular stress response is important for maintaining function and survival, overactivation of late-stage stress effectors cause dysfunction and death. We show that the myelin transcription factors (TFs) Myt1 (Nzf2), Myt2 (Myt1l, Nztf1, and Png-1), and Myt3 (St18 and Nzf3) prevent such overactivation in islet ß cells. Thus, we found that co-inactivating the Myt TFs in mouse pancreatic progenitors compromised postnatal ß cell function, proliferation, and survival, preceded by upregulation of late-stage stress-response genes activating transcription factors (e.g., Atf4) and heat-shock proteins (Hsps). Myt1 binds putative enhancers of Atf4 and Hsps, whose overexpression largely recapitulated the Myt-mutant phenotypes. Moreover, Myt(MYT)-TF levels were upregulated in mouse and human ß cells during metabolic stress-induced compensation but downregulated in dysfunctional type 2 diabetic (T2D) human ß cells. Lastly, MYT knockdown caused stress-gene overactivation and death in human EndoC-ßH1 cells. These findings suggest that Myt TFs are essential restrictors of stress-response overactivity.

Neurog3-Independent Methylation Is the Earliest Detectable Mark Distinguishing Pancreatic Progenitor Identity.

In the developing pancreas, transient Neurog3-expressing progenitors give rise to four major islet cell types: α, ß, δ, and γ; when and how the Neurog3+ cells choose cell fate is unknown. Using single-cell RNA-seq, trajectory analysis, and combinatorial lineage tracing, we showed here that the Neurog3+ cells co-expressing Myt1 (i.e., Myt1+Neurog3+) were biased toward ß cell fate, while those not simultaneously expressing Myt1 (Myt1-Neurog3+) favored α fate. Myt1 manipulation only marginally affected α versus ß cell specification, suggesting Myt1 as a marker but not determinant for islet-cell-type specification. The Myt1+Neurog3+ cells displayed higher Dnmt1 expression and enhancer methylation at Arx, an α-fate-promoting gene. Inhibiting Dnmts in pancreatic progenitors promoted α cell specification, while Dnmt1 overexpression or Arx enhancer hypermethylation favored ß cell production. Moreover, the pancreatic progenitors contained distinct Arx enhancer methylation states without transcriptionally definable sub-populations, a phenotype independent of Neurog3 activity. These data suggest that Neurog3-independent methylation on fate-determining gene enhancers specifies distinct endocrine-cell programs.

Synaptotagmin 4 Regulates Pancreatic ß Cell Maturation by Modulating the Ca2+ Sensitivity of Insulin Secretion Vesicles.

Islet ß cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated insulin secretion (GSIS). Here we show that ß cells of newborns secrete more insulin than adults in response to similar intracellular Ca2+ concentrations, suggesting differences in the Ca2+ sensitivity of insulin secretion. Synaptotagmin 4 (Syt4), a non-Ca2+ binding paralog of the ß cell Ca2+ sensor Syt7, increased by ∼8-fold during ß cell maturation. Syt4 ablation increased basal insulin secretion and compromised GSIS. Precocious Syt4 expression repressed basal insulin secretion but also impaired islet morphogenesis and GSIS. Syt4 was localized on insulin granules and Syt4 levels inversely related to the number of readily releasable vesicles. Thus, transcriptional regulation of Syt4 affects insulin secretion; Syt4 expression is regulated in part by Myt transcription factors, which repress Syt4 transcription. Finally, human SYT4 regulated GSIS in EndoC-ßH1 cells, a human ß cell line. These findings reveal the role that altered Ca2+ sensing plays in regulating ß cell maturation.

Effects of Electro-Acupuncture at Zusanli, Guanyuan for Sepsis Patients and Its Mechanism through Immune Regulation.

OBJECTIVE: To evaluate the effect of electro-acupuncture on Zusanli (ST 36), Guanyuan (RN 4) in patients with sepsis, and explore its mechanism in term of immune regulation. METHODS: In this prospective randomized controlled trial, 60 patients with sepsis were randomly assigned to the control group and the intervention group equally by block randomization. Patients in the control group received routine treatment and those in the intervention group received electro-acupuncture at bilateral Zusanli and Guanyuan in addition to routine treatment, respectively. The mortality at 28 days, Acute Physiology and Chronic Health Evaluation (APACHE)-II score were compared to evaluate the effect, and the levels of T cell subsets (CD3+, CD4+, CD8+, CD4+/CD8+) and monocytes of human leukocyte antigen (HLA)-DR using flow cytometry were compared to explore the mechanism of this combined treatment. RESULTS: Fifty-eight patients completed the trial with 29 in each group. There was no significant difference of mortality in the 28th day between the two groups, with 5 death of 29 patients in the intervention group (17.2%) and 9 of 29 in the control group (31.0%). After treatment, APACHE-II score of both groups was significantly decreased, however, score of the intervention group was lower than the control group (13.28±7.07 vs. 17.10±5.83; P<0.01). The levels of CD3+, CD4+, CD8+ and CD4+/CD8+ ratio of the intervention group improved after treatment and were higher than the control group (59.71%±11.94% vs. 52.54%±11.86%; 36.46%±7.60% vs. 31.58%±10.23%; 18.40%±8.82% vs. 23.07%±7.30%; 2.38±1.14 vs. 1.54±0.80, respectively; all P<0.05). The expression of HLA-DR significantly increased after treatment in the intervention group than that in the control group (7.28%±9.26% vs. 1.27%±7.00%; P<0.01). CONCLUSION: Electro-acupuncture at Zusanli and Guanyuan could improve clinical curative effect in patients with sepsis, which might be achieved by regulation of the immune system.

Microtubules Negatively Regulate Insulin Secretion in Pancreatic ß Cells.

For glucose-stimulated insulin secretion (GSIS), insulin granules have to be localized close to the plasma membrane. The role of microtubule-dependent transport in granule positioning and GSIS has been debated. Here, we report that microtubules, counterintuitively, restrict granule availability for secretion. In ß cells, microtubules originate at the Golgi and form a dense non-radial meshwork. Non-directional transport along these microtubules limits granule dwelling at the cell periphery, restricting granule availability for secretion. High glucose destabilizes microtubules, decreasing their density; such local microtubule depolymerization is necessary for GSIS, likely because granule withdrawal from the cell periphery becomes inefficient. Consistently, microtubule depolymerization by nocodazole blocks granule withdrawal, increases their concentration at exocytic sites, and dramatically enhances GSIS in vitro and in mice. Furthermore, glucose-driven MT destabilization is balanced by new microtubule formation, which likely prevents over-secretion. Importantly, microtubule density is greater in dysfunctional ß cells of diabetic mice.

Identification of a novel mouse P4-ATPase family member highly expressed during spermatogenesis.

P4-ATPases are transmembrane proteins unique to eukaryotes that play a fundamental role in vesicular transport. They have been proposed to act as phospholipid flippases thereby regulating lipid topology in cellular membranes. We cloned and characterized a novel murine P4-ATPase that is specifically expressed in testis, and named it FetA (flippase expressed in testis splicing form A). When expressed in Saccharomyces cerevisiae, FetA localizes partially to the plasma membrane resulting in increased internalization of NBD-labeled phosphatidylethanolamine and phosphatidylcholine, supporting a role for FetA in the inward lipid translocation across cellular membranes. In mouse testis, FetA protein is detected in gamete cells, from pachytene spermatocytes to mature sperms, and its intracellular localization is tightly related with acrosome formation, a process that involves intensive intracellular vesicle formation and fusion. Furthermore, loss-of-function of FetA by RNA interference in mastocytoma P815 cells profoundly perturbs the structural organization of the Golgi complex and causes loss of constitutive secretion at lower temperature. Our findings point to an essential role of FetA in Golgi morphology and secretory function, suggesting a crucial role for this novel murine P4-ATPase in spermatogenesis.

Smad6 inhibits the transcriptional activity of Tbx6 by mediating its degradation.

Members of the bone morphogenetic protein (BMP) and T-box gene families play several critical roles in the early embryonic development and tissue homeostasis. Although BMP proteins are the upstream regulators of T-box genes, few studies have investigated the molecular mechanisms between these two protein families. Here, we report that Tbx6 interacts directly with Smad6, an inhibitory Smad that antagonizes the BMP signal. This interaction is mediated through the Mad homology 2 (MH2) domain of Smad6 and residues 90-180 of Tbx6. We demonstrate that Smad6 facilitates the degradation of Tbx6 protein through recruitment of Smurf1, a ubiquitin E3 ligase. Consequently, Smad6 reduces Tbx6-mediated Myf-5 gene activation. Furthermore, specific knockdown of endogenous Smad6 and Smurf1 by small interfering RNA increases the protein levels of Tbx6 and enhance the expression of Tbx6 target genes. Collectively, these findings reveal that Smad6 serves as a critical mediator of BMP signal via a functional interaction with Tbx6, thus regulating the activation of Tbx6 downstream genes during cell differentiation.

Xenopus Tbx6 mediates posterior patterning via activation of Wnt and FGF signalling.

In vertebrates, the patterning of anterior-posterior (AP) axis is a fundamental process during embryogenesis. Wnt and FGF signalling pathways play important roles in regulating the patterning of embryo AP axis. Mouse Tbx6 encodes a transcription factor that has been demonstrated to be involved in the specification of the posterior tissue in mouse embryonic body. Here, we prove that morpholino-induced knockdown of XTbx6 impairs posterior development, indicating the requirement of XTbx6 in this process. Meanwhile, gain of XTbx6 function is sufficient to induce ectopic posterior structures in Xenopus embryos. Furthermore, XTbx6 activates the expression of Xwnt8 and FGF8, which are two mediators of posterior development, suggesting a mechanism by which XTbx6 modulates posterior patterning via Wnt and FGF signalling pathway activation.

Optimized adaptor polymerase chain reaction method for efficient genomic walking.

Genomic walking is one of the most useful approaches in genome-related research. Three kinds of PCR-based methods are available for this purpose. However, none of them has been generally applied because they are either insensitive or inefficient. Here we present an efficient PCR protocol, an optimized adaptor PCR method for genomic walking. Using a combination of a touchdown PCR program and a special adaptor, the optimized adaptor PCR protocol achieves high sensitivity with low background noise. By applying this protocol, the insertion sites of a gene trap mouse line and two gene promoters from the incompletely sequenced Xenopus laevis genome were successfully identified with high efficiency. The general application of this protocol in genomic walking was promising.

The role of Paraxial Protocadherin in Xenopus otic placode development.

Vertebrate inner ear develops from its rudiment, otic placode, which later forms otic vesicle and gives rise to tissues comprising the entire inner ear. Although several signaling molecules have been identified as candidates responsible for inner ear specification and patterning, many details remain elusive. Here, we report that Paraxial Protocadherin (PAPC) is required for otic vesicle formation in Xenopus embryos. PAPC is expressed strictly in presumptive otic placode and later in otic vesicle during inner ear morphogenesis. Knockdown of PAPC by dominant-negative PAPC results in the failure of otic vesicle formation and the loss of early inner ear markers Sox9 and Tbx2, suggesting the requirement of PAPC in the early stage of otic vesicle development. However, PAPC alone is not sufficient to induce otic placode formation.

A novel spermatogenesis-specific uPAR gene expressed in human and mouse testis.

The urokinase receptor, uPAR, which binds to the urinary-type plasminogen activator, controls matrix degradation in the processes of tissue remodeling, cell migration, and invasion. In the present study, we found a new urokinase receptor gene that encodes a 249-amino acid putative protein. Northern blot analysis showed specific expression in the testis of this gene, which we named the spermatogenesis-related gene (SGRG). In situ hybridization revealed a strong expression signal for SGRG in spermatogonia, but not in spermatocytes. Therefore, we conjecture that SGRG may regulate spermatocyte migration through breakdown of extracellular matrix protein barriers in spermatogenesis. Since SGRG is specifically expressed in spermatogonia, it provides an attractive candidate for development of a contraceptive vaccine.

[Progress in the investigation of Avian sex determination and sex identification].

Avian sex determination is a multiple gene regulation cascade. Genes such as the Z chromosome-linked DMRT1 gene, W chromosome-linked PKCIW gene and other factors have been demonstrated to be involved in this process. In this paper, we review the recent progress in this field. The investigation of functions of sex determinate genes and methods of sexing identification in birds are also discussed.