The weaning process concerns all patients receiving mechanical ventilation. A previous classification into simple, prolonged, and difficult weaning ignored weaning failure and presupposed the use of ...spontaneous breathing trials.
To describe the weaning process, defined as starting with any attempt at separation from mechanical ventilation and its prognosis, according to a new operational classification working for all patients under ventilation.
This was a multinational prospective multicenter observational study over 3 months of all patients receiving mechanical ventilation in 36 intensive care units, with daily collection of ventilation and weaning modalities. Pragmatic definitions of separation attempt and weaning success allowed us to allocate patients in four groups.
A total of 2,729 patients were enrolled. Although half of them could not be classified using the previous definition, 99% entered the groups on the basis of our new definition as follows: 24% never started a weaning process, 57% had a weaning process of less than 24 hours (group 1), 10% had a difficult weaning of more than 1 day and less than 1 week (group 2), and 9% had a prolonged weaning duration of 1 week or more (group 3). Duration of ventilation, intensive care unit stay, and mortality (6, 17, and 29% for the three groups, respectively) all significantly increased from one group to the next. The unadjusted risk of dying was 19% after the first separation attempt and increased to 37% after 10 days.
A new classification allows us to categorize all weaning situations. Every additional day without a weaning success after the first separation attempt increases the risk of dying.
Insulin-secreting pancreatic β-cells play a critical role in blood glucose homeostasis and the development of type 2 diabetes (T2D) in the context of insulin resistance. Based on data obtained at the ...whole cell level using poorly specific chemical probes, reactive oxygen species (ROS) such as superoxide and hydrogen peroxide have been proposed to contribute to the stimulation of insulin secretion by nutrients (positive role) and to the alterations of cell survival and secretory function in T2D (negative role). This raised the controversial hypothesis that any attempt to decrease β-cell oxidative stress and apoptosis in T2D would further impair insulin secretion. Over the last decade, the development of genetically-encoded redox probes that can be targeted to cellular compartments of interest and are specific of redox couples allowed the evaluation of short- and long-term effects of nutrients on β-cell redox changes at the subcellular level. The data indicated that the nutrient regulation of β-cell redox signaling and ROS toxicity is far more complex than previously thought and that the subcellular compartmentation of these processes cannot be neglected when evaluating the mechanisms of ROS production or the efficacy of antioxidant enzymes and antioxidant drugs under glucolipotoxic conditions and in T2D. In this review, we present what is currently known about the compartmentation of redox homeostatic systems and tools to investigate it. We then review data about the effects of nutrients on β-cell subcellular redox state under normal conditions and in the context of T2D and discuss challenges and opportunities in the field.
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•New genetically-encoded redox probes allow the study of changes in cellular redox state in a compartment-specific manner.•Nutrient-induced changes in pancreatic β-cell redox state are highly compartmentalized.•The impact of acute glucose stimulation on cytosolic and mitochondrial redox probe oxidation remains controversial.•The cytosol, mitochondria, ER and peroxisomes could play a role in β-cell oxidative stress under nutrient toxic conditions.•Intravital microscopy may improve the study of subcellular oxidative stress in insulin resistance and type 2 diabetes.
We are aware, and agree, that the term “oxidants” may be more adequate than “reactive oxygen species” (ROS) to collectively refer to hydrogen peroxide (H2O2), superoxide (O2−), and other oxidants when the specific molecule involved is not known 1. However, to avoid disturbing the reader from the β-cell field, and as both terms are equally vague, we chose to use “ROS” rather than “oxidants” throughout this review.
Scope
Aspalathin, the main polyphenolic phytochemical of rooibos (Aspalathus linearis), has been attributed with health promoting properties, including a glucose lowering effect that can prove ...interesting for application as nutraceutical or therapeutic in (pre‐)diabetics. Preservation of β cell mass in the pancreas is considered a key issue for diabetes prevention or treatment, therefore the aim is to investigate whether aspalathin also has β cell cytoprotective potential.
Methods and results
Rat pancreatic islets and the β cell line Insulinoma 1E (INS1E) are studied in vitro after exposure to various cytotoxic agents, namely streptozotocin (STZ), hydrogen peroxide, or chronic high glucose. The effect of aspalathin on cell survival and apoptosis is studied. Expression of relevant cytoprotective genes is analyzed by qRT‐PCR and proteins by Western blot. Aspalathin is found to protect β cells against cytotoxicity and apoptosis. This is associated with increased translocation of nuclear factor erythroid 2‐related factor 2 (NRF2) and expression of its antioxidant target genes heme oxygenase 1 (Hmox1), NAD(P)H quinone dehydrogenase 1 (Nqo‐1), and superoxide dismutase 1 (Sod1).
Conclusion
It is proposed that aspalathin protects β cells against glucotoxicity and oxidative stress by increasing the expression of NRF2‐regulated antioxidant enzymes. This indicates that aspalathin is an interesting β cell cytoprotectant.
Aspalathin, the major antioxidative component of rooibos tea, has antidiabetic potential. The present study shows that aspalathin protects β cells in vitro against oxidative damage. It proposes the interaction of aspalathin with KEAP1 and P62 proteins leading to accumulation of NRF2 in the nucleus and activation of the transcription of anti‐oxidant protective genes like Hmox1, Nqo1, and Sod1.
► Hyperglycemia exert “toxic” effects on the β-cell phenotype in type 2 diabetes. ► β-Cell “glucotoxicity” involves complex cellular and molecular mechanisms. ► ROS, UPR and loss of differentiation ...play major roles in β-cell glucotoxicity. ► There is an emerging role of AGEs and hypoxia in β-cell glucotoxicity. ► Manipulation of these mechanisms is protective in vitro and in animal models.
It is well established that regular physiological stimulation by glucose plays a crucial role in the maintenance of the β-cell differentiated phenotype. In contrast, prolonged or repeated exposure to elevated glucose concentrations both in vitro and in vivo exerts deleterious or toxic effects on the β-cell phenotype, a concept termed as glucotoxicity.
Evidence indicates that the latter may greatly contribute to the pathogenesis of type 2 diabetes. Through the activation of several mechanisms and signaling pathways, high glucose levels exert deleterious effects on β-cell function and survival and thereby, lead to the worsening of the disease over time. While the role of high glucose-induced β-cell overstimulation, oxidative stress, excessive Unfolded Protein Response (UPR) activation, and loss of differentiation in the alteration of the β-cell phenotype is well ascertained, at least in vitro and in animal models of type 2 diabetes, the role of other mechanisms such as inflammation, O-GlcNacylation, PKC activation, and amyloidogenesis requires further confirmation. On the other hand, protein glycation is an emerging mechanism that may play an important role in the glucotoxic deterioration of the β-cell phenotype. Finally, our recent evidence suggests that hypoxia may also be a new mechanism of β-cell glucotoxicity.
Deciphering these molecular mechanisms of β-cell glucotoxicity is a mandatory first step toward the development of therapeutic strategies to protect β-cells and improve the functional β-cell mass in type 2 diabetes.
Aims/hypothesis
The mechanisms responsible for beta cell compensation in obesity and for beta cell failure in type 2 diabetes are poorly defined. The mRNA levels of several metallothionein (MT) genes ...are upregulated in islets from individuals with type 2 diabetes, but their role in beta cells is not clear. Here we examined: (1) the temporal changes of islet
Mt1
and
Mt2
gene expression in mouse models of beta cell compensation and failure; and (2) the role of
Mt1
and
Mt2
in beta cell function and glucose homeostasis in mice.
Methods
Mt1
and
Mt2
expression was assessed in islets from: (1) control lean (chow diet-fed) and diet-induced obese (high-fat diet-fed for 6 weeks) mice; (2) mouse models of diabetes (
db/db
mice) at 6 weeks old (prediabetes) and 16 weeks old (after diabetes onset) and age-matched
db/+
(control) mice; and (3) obese non-diabetic
ob/ob
mice (16-week-old) and age-matched
ob/+
(control) mice.
MT1E
,
MT1X
and
MT2A
expression was assessed in islets from humans with and without type 2 diabetes.
Mt1-Mt2
double-knockout (KO) mice, transgenic mice overexpressing
Mt1
under the control of its natural promoter (Tg-
Mt1
) and corresponding control mice were also studied. In MIN6 cells, MT1 and MT2 were inhibited by small interfering RNAs. mRNA levels were assessed by real-time RT-PCR, plasma insulin and islet MT levels by ELISA, glucose tolerance by i.p. glucose tolerance tests and overnight fasting-1 h refeeding tests, insulin tolerance by i.p
.
insulin tolerance tests, insulin secretion by RIA, cytosolic free Ca
2+
concentration with Fura-2 leakage resistant (Fura-2 LR), cytosolic free Zn
2+
concentration with Fluozin-3, and NAD(P)H by autofluorescence.
Results
Mt1
and
Mt2
mRNA levels were reduced in islets of murine models of beta cell compensation, whereas they were increased in diabetic
db/db
mice. In humans,
MT1X
mRNA levels were significantly upregulated in islets from individuals with type 2 diabetes in comparison with non-diabetic donors, while
MT1E
and
MT2A
mRNA levels were unchanged. Ex vivo, islet
Mt1
and
Mt2
mRNA and MT1 and MT2 protein levels were downregulated after culture with glucose at 10–30 mmol/l vs 2–5 mmol/l, in association with increased insulin secretion. In human islets, mRNA levels of
MT1E
,
MT1X
and
MT2A
were downregulated by stimulation with physiological and supraphysiological levels of glucose. In comparison with wild-type (WT) mice,
Mt1-Mt2
double-KO mice displayed improved glucose tolerance in association with increased insulin levels and enhanced insulin release from isolated islets. In contrast, isolated islets from Tg-
Mt1
mice displayed impaired glucose-stimulated insulin secretion (GSIS). In both
Mt1-Mt2
double-KO and Tg-
Mt1
models, the changes in GSIS occurred despite similar islet insulin content, rises in cytosolic free Ca
2+
concentration and NAD(P)H levels, or intracellular Zn
2+
concentration vs WT mice. In MIN6 cells, knockdown of MT1 but not MT2 potentiated GSIS, suggesting that
Mt1
rather than
Mt2
affects beta cell function.
Conclusions/interpretation
These findings implicate
Mt1
as a negative regulator of insulin secretion. The downregulation of
Mt1
is associated with beta cell compensation in obesity, whereas increased
Mt1
accompanies beta cell failure and type 2 diabetes.
Therapy resistance arises from heterogeneous drug-tolerant persister cells or minimal residual disease (MRD) through genetic and nongenetic mechanisms. A key question is whether specific molecular ...features of the MRD ecosystem determine which of these two distinct trajectories will eventually prevail. We show that, in melanoma exposed to mitogen-activated protein kinase therapeutics, emergence of a transient neural crest stem cell (NCSC) population in MRD concurs with the development of nongenetic resistance. This increase relies on a glial cell line-derived neurotrophic factor-dependent signaling cascade, which activates the AKT survival pathway in a focal adhesion kinase (FAK)-dependent manner. Ablation of the NCSC population through FAK inhibition delays relapse in patient-derived tumor xenografts. Strikingly, all tumors that ultimately escape this treatment exhibit resistance-conferring genetic alterations and increased sensitivity to extracellular signal-regulated kinase inhibition. These findings identify an approach that abrogates the nongenetic resistance trajectory in melanoma and demonstrate that the cellular composition of MRD deterministically imposes distinct drug resistance evolutionary paths.
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•Tumors recurrently select either a genetic or nongenetic drug resistance trajectory•FAK signaling is activated in melanoma drug persisters with a neural crest-like state•Targeting neural crest-like cells prevents nongenetic drug resistance evolution•The cellular composition of MRD dictates the evolutionary path to resistance
Marin-Bejar et al. identify focal adhesion kinase (FAK) as a vulnerability of melanoma drug persisters harboring a neural crest-like state. Targeting these cells, using a FAK inhibitor, prevents the development of nongenetic, but not genetic, resistance, indicating that the path to resistance is dictated by the cellular composition of minimal residual disease.
The mechanisms underpinning beta‐cell compensation for obesity‐associated insulin resistance and beta‐cell failure in type 2 diabetes remain poorly understood. We used a large‐scale strategy to ...determine the time‐dependent transcriptomic changes in islets of diabetes‐prone db/db and diabetes‐resistant ob/ob mice at 6 and 16 weeks of age. Differentially expressed genes were subjected to cluster, gene ontology, pathway and gene set enrichment analyses. A distinctive gene expression pattern was observed in 16 week db/db islets in comparison to the other groups with alterations in transcriptional regulators of islet cell identity, upregulation of glucose/lipid metabolism, and various stress response genes, and downregulation of specific amino acid transport and metabolism genes. In contrast, ob/ob islets displayed a coordinated downregulation of metabolic and stress response genes at 6 weeks of age, suggestive of a preemptive reconfiguration in these islets to lower the threshold of metabolic activation in response to increased insulin demand thereby preserving beta‐cell function and preventing cellular stress. In addition, amino acid transport and metabolism genes were upregulated in ob/ob islets, suggesting an important role of glutamate metabolism in beta‐cell compensation. Gene set enrichment analysis of differentially expressed genes identified the enrichment of binding motifs for transcription factors, FOXO4, NFATC1, and MAZ. siRNA‐mediated knockdown of these genes in MIN6 cells altered cell death, insulin secretion, and stress gene expression. In conclusion, these data revealed novel gene regulatory networks involved in beta‐cell compensation and failure. Preemptive metabolic reconfiguration in diabetes‐resistant islets may dampen metabolic activation and cellular stress during obesity.
Whereas significant anti-tumor responses are observed in most BRAF
-mutant melanoma patients exposed to MAPK-targeting agents, resistance almost invariably develops. Here, we show that in ...therapy-responsive cells BRAF inhibition induces downregulation of the processing of Sterol Regulator Element Binding (SREBP-1) and thereby lipogenesis. Irrespective of the escape mechanism, therapy-resistant cells invariably restore this process to promote lipid saturation and protect melanoma from ROS-induced damage and lipid peroxidation. Importantly, pharmacological SREBP-1 inhibition sensitizes BRAF
-mutant therapy-resistant melanoma to BRAF
inhibitors both in vitro and in a pre-clinical PDX in vivo model. Together, these data indicate that targeting SREBP-1-induced lipogenesis may offer a new avenue to overcome acquisition of resistance to BRAF-targeted therapy. This work also provides evidence that targeting vulnerabilities downstream of oncogenic signaling offers new possibilities in overcoming resistance to targeted therapies.
The loss of functional beta cell mass characterises all forms of diabetes. Beta cells are highly susceptible to stress, including cytokine, endoplasmic reticulum (ER) and oxidative stress. This study ...examined the role of pleckstrin homology-like, domain family A, member 3 (Phlda3) in beta cell survival under stress conditions and the regulatory basis. We found that the mRNA levels of Phlda3 were markedly upregulated in vivo in the islets of diabetic humans and mice. In vitro, exposure of MIN6 cells or islets to cytokines, palmitate, thapsigargin or ribose upregulated Phlda3 mRNA and protein levels, concurrent with the induction of ER stress (Ddit3 and Trb3) and antioxidant (Hmox1) genes. Furthermore, H
O
treatment markedly increased PHLDA3 immunostaining in human islets. Phlda3 expression was differentially regulated by adaptive (Xbp1) and apoptotic (Ddit3) unfolded protein response (UPR) mediators. siRNA-mediated knockdown of Xbp1 inhibited the induction of Phlda3 by cytokines and palmitate, whereas knockdown of Ddit3 upregulated Phlda3. Moreover, knockdown of Phlda3 potentiated cytokine-induced apoptosis in association with upregulation of inflammatory genes (iNos, IL1β and IκBα) and NFκB phosphorylation and downregulation of antioxidant (Gpx1 and Srxn1) and adaptive UPR (Xbp1, Hspa5 and Fkbp11) genes. Knockdown of Phlda3 also potentiated apoptosis under oxidative stress conditions induced by ribose treatment. These findings suggest that Phlda3 is crucial for beta cell survival under stress conditions. Phlda3 regulates the cytokine, oxidative and ER stress responses in beta cells via the repression of inflammatory gene expression and the maintenance of antioxidant and adaptive UPR gene expression. Phlda3 may promote beta cell survival in diabetes.
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