The role of lipid droplet associated proteins in inherited human disorders.

Proteins which associate with the surface of lipid droplets are intimately involved in the regulation of the droplets. Several human inherited disorders have now been linked to loss- and, in some cases, likely gain-of-function mutations in the genes encoding these proteins. These are summarised in this Graphical Review.

Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance.

The failure of metabolic tissues to appropriately respond to insulin ("insulin resistance") is an early marker in the pathogenesis of type 2 diabetes. Protein phosphorylation is central to the adipocyte insulin response, but how adipocyte signaling networks are dysregulated upon insulin resistance is unknown. Here we employ phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults causing insulin resistance, we observe a marked rewiring of the insulin signaling network. This includes both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying dysregulated phosphosites common to multiple insults reveals subnetworks containing non-canonical regulators of insulin action, such as MARK2/3, and causal drivers of insulin resistance. The presence of several bona fide GSK3 substrates among these phosphosites led us to establish a pipeline for identifying context-specific kinase substrates, revealing widespread dysregulation of GSK3 signaling. Pharmacological inhibition of GSK3 partially reverses insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that includes dysregulated MARK2/3 and GSK3 activity.

A mouse model of human mitofusin-2-related lipodystrophy exhibits adipose-specific mitochondrial stress and reduced leptin secretion.

Mitochondrial dysfunction has been reported in obesity and insulin resistance, but primary genetic mitochondrial dysfunction is generally not associated with these, arguing against a straightforward causal relationship. A rare exception, recently identified in humans, is a syndrome of lower body adipose loss, leptin-deficient severe upper body adipose overgrowth, and insulin resistance caused by the p.Arg707Trp mutation in MFN2, encoding mitofusin 2. How the resulting selective form of mitochondrial dysfunction leads to tissue- and adipose depot-specific growth abnormalities and systemic biochemical perturbation is unknown. To address this, Mfn2R707W/R707W knock-in mice were generated and phenotyped on chow and high fat diets. Electron microscopy revealed adipose-specific mitochondrial morphological abnormalities. Oxidative phosphorylation measured in isolated mitochondria was unperturbed, but the cellular integrated stress response was activated in adipose tissue. Fat mass and distribution, body weight, and systemic glucose and lipid metabolism were unchanged, however serum leptin and adiponectin concentrations, and their secretion from adipose explants were reduced. Pharmacological induction of the integrated stress response in wild-type adipocytes also reduced secretion of leptin and adiponectin, suggesting an explanation for the in vivo findings. These data suggest that the p.Arg707Trp MFN2 mutation selectively perturbs mitochondrial morphology and activates the integrated stress response in adipose tissue. In mice, this does not disrupt most adipocyte functions or systemic metabolism, whereas in humans it is associated with pathological adipose remodelling and metabolic disease. In both species, disproportionate effects on leptin secretion may relate to cell autonomous induction of the integrated stress response.

A high-content endogenous GLUT4 trafficking assay reveals new aspects of adipocyte biology.

Insulin-induced GLUT4 translocation to the plasma membrane in muscle and adipocytes is crucial for whole-body glucose homeostasis. Currently, GLUT4 trafficking assays rely on overexpression of tagged GLUT4. Here we describe a high-content imaging platform for studying endogenous GLUT4 translocation in intact adipocytes. This method enables high fidelity analysis of GLUT4 responses to specific perturbations, multiplexing of other trafficking proteins and other features including lipid droplet morphology. Using this multiplexed approach we showed that Vps45 and Rab14 are selective regulators of GLUT4, but Trarg1, Stx6, Stx16, Tbc1d4 and Rab10 knockdown affected both GLUT4 and TfR translocation. Thus, GLUT4 and TfR translocation machinery likely have some overlap upon insulin-stimulation. In addition, we identified Kif13A, a Rab10 binding molecular motor, as a novel regulator of GLUT4 traffic. Finally, comparison of endogenous to overexpressed GLUT4 highlights that the endogenous GLUT4 methodology has an enhanced sensitivity to genetic perturbations and emphasises the advantage of studying endogenous protein trafficking for drug discovery and genetic analysis of insulin action in relevant cell types.

Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate.

Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of murine TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. These data place TRARG1 within the insulin signaling network and provide insights into how GSK3 regulates GLUT4 trafficking in adipocytes.

Lactate production is a prioritized feature of adipocyte metabolism.

Adipose tissue is essential for whole-body glucose homeostasis, with a primary role in lipid storage. It has been previously observed that lactate production is also an important metabolic feature of adipocytes, but its relationship to adipose and whole-body glucose disposal remains unclear. Therefore, using a combination of metabolic labeling techniques, here we closely examined lactate production of cultured and primary mammalian adipocytes. Insulin treatment increased glucose uptake and conversion to lactate, with the latter responding more to insulin than did other metabolic fates of glucose. However, lactate production did not just serve as a mechanism to dispose of excess glucose, because we also observed that lactate production in adipocytes did not solely depend on glucose availability and even occurred independently of glucose metabolism. This suggests that lactate production is prioritized in adipocytes. Furthermore, knocking down lactate dehydrogenase specifically in the fat body of Drosophila flies lowered circulating lactate and improved whole-body glucose disposal. These results emphasize that lactate production is an additional metabolic role of adipose tissue beyond lipid storage and release.

Phosphoproteomics of Acute Cell Stressors Targeting Exercise Signaling Networks Reveal Drug Interactions Regulating Protein Secretion.

Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of ß-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.

Sevoflurane postconditioning improves the spatial learning and memory impairments induced by hemorrhagic shock and resuscitation through suppressing IRE1α-caspase-12-mediated endoplasmic reticulum stress pathway.

Severe hemorrhagic shock induces cognitive dysfunction by promoting cell death mediated by activating endoplasmic reticulum (ER) stress. Sevoflurane postconditioning prevents neuronal apoptosis against cerebral ischemia/reperfusion injury. It is unknown if this protective effect on hemorrhagic shock and resuscitation rats (HSR) is associated with ER stress attenuation. Male adult Sprague-Dawley rats were subjected HSR by removing 40% blood volume within 30 min, and 60 min later the animals were resuscitated with infusion of the removing blood in 30 min. Sevoflurane postconditioning was performed by inhaling sevoflurane at three different concentrations (0.5, 1.0, 1.5 MAC) at the onset of resuscitation for 30 min. Severe hypotension (mean arterial pressure 40-45 mmHg) occurred in the shock session for 60 min accompanying with significantly elevated lactate, decreased BE and pH values in arterial blood gas analysis. There were impaired spatial learning and memory following HSR indicated by persistently longer escape latency and lower correct rate, as well as less duration and crossing in the target quadrant by using Morris water maze and Y-maze tests. In the hippocampal CA1 region, there was significantly higher activity of caspase-3 induced by HSR. HSR also elevated the expression of inositol-requiring enzyme 1α (IRE1α) and caspase-12 in the hippocampus by western blot analysis. Sevoflurane postconditioning at 1.0 and 1.5 MAC significantly reversed these changes. These findings suggested that sevoflurane postconditioning could improve spatial learning and memory deficits induced by severe hemorrhagic shock and subsequent resuscitation. The suppression of endoplasmic reticulum stress provided critical contribution in neural apoptosis mediated by IRE1α-caspase-12 pathway.

Postconditioning with sevoflurane ameliorates spatial learning and memory deficit via attenuating endoplasmic reticulum stress induced neuron apoptosis in a rat model of hemorrhage shock and resuscitation.

Hemorrhage shock could initiate endoplasmic reticulum stress (ERS) and then induce neuronal apoptosis. The aim of this study was to investigate whether sevoflurane postconditioning could attenuate brain injury via suppressing apoptosis induced by ERS. Seventy male rats were randomized into five groups: sham, shock, low concentration (sevo1, 1.2%), middle concentration (sevo2, 2.4%) and high concentration (sevo3, 3.6%) of sevoflurane postconditioning. Hemorrhage shock was induced by removing 40% of the total blood volume during an interval of 30 min. 1 h after the completion of bleeding, the animals were reinfused with shed blood during the ensuing 30 min. The spatial learning and memory ability of rats were measured by Morris water maze (MWM) test three days after the operation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells in the hippocampus CA1 region were assessed after the MWM test. The expression of C/EBP-homologousprotein (CHOP) and glucose-regulated protein 78 (GRP78) in the hippocampus were measured at 24 h after reperfusion. We found that sevoflurane postconditioning with the concentrations of 2.4% and 3.6% significantly ameliorated the spatial learning and memory ability, decreased the TUNEL-positive cells, and reduced the GRP78 and CHOP expression compared with the shock group. These results suggested that sevoflurane postconditioning with the concentrations of 2.4% and 3.6% could ameliorate spatial learning and memory deficit after hemorrhage shock and resuscitation injury via suppressing apoptosis induced by ERS.

Membrane Topology of Trafficking Regulator of GLUT4 1 (TRARG1).

Trafficking regulator of GLUT4 1 (TRARG1) was recently identified to localize to glucose transporter type 4 (GLUT4) storage vesicles (GSVs) and to positively regulate GLUT4 trafficking. Our knowledge of TRARG1 structure and membrane topology is limited to predictive models, hampering efforts to further our mechanistic understanding of how it carries out its functions. Here, we use a combination of bioinformatics prediction tools and biochemical assays to define the membrane topology of the 173-amino acid mouse TRARG1. These analyses revealed that, contrary to the consensus prediction, the N-terminus is cytosolic and that a short segment at the C-terminus resides in the luminal/extracellular space. Based on our biochemical analyses including membrane association and antibody accessibility assays, we conclude that TRARG1 has one transmembrane domain (TMD) (145-172) and a re-entrant loop between residues 101 and 127.

Postconditioning with sevoflurane ameliorates spatial learning and memory deficit after hemorrhage shock and resuscitation in rats.

BACKGROUND: Severe hemorrhage shock and resuscitation are a systemic ischemia-reperfusion phenomenon which can induce learning and memory deficit in human and rats. Sevoflurane postconditioning has been proved to offer neuroprotection under different setting of cerebral ischemia-reperfusion in rats. The aim of this study was to investigate whether sevoflurane postconditioning could improve spatial learning and memory ability after hemorrhage shock and resuscitation in rats. METHODS: Thirty-five male rats were randomized into five groups: sham group, shock group, low concentration (sevo1, 1.2%), middle concentration (sevo2, 2.4%), and high concentration (sevo3, 3.6%) of sevoflurane postconditioning groups. The spatial learning and memory ability of rats were measured by Morris water maze 3 d after the operation. The expression of choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE) in the hippocampus CA1 region was observed by immunohistochemistry method after the Morris water maze test. RESULTS: The ability of spatial learning and memory of rats and the expression of CHAT was significantly declined, while the expression of ACHE increased in the shock group compared with the sham group (P < 0.05). Sevoflurane postconditioning with the concentrations of 2.4% and 3.6% significantly ameliorated the spatial learning and memory ability and increased the expression of CHAT and decreased the expression of ACHE in hippocampal CA1 region when compared with shock group (P < 0.05). CONCLUSIONS: Postconditioning with sevoflurane at the concentrations of 2.4% and 3.6% which improved the ability of spatial learning and memory after hemorrhage shock and resuscitation in rats may involve the protection of the cholinergic neurons in hippocampal CA1 region.

Self-Fluid Management in Prevention of Kidney Stones: A PRISMA-Compliant Systematic Review and Dose-Response Meta-Analysis of Observational Studies.

Epidemiologic studies have suggested that daily fluid intake that achieves at least 2.5 L of urine output per day is protective against kidney stones. However, the precise quantitative nature of the association between fluid intake and kidney stone risk, as well as the effect of specific types of fluids on such risk, are not entirely clear.We conducted a systematic review and dose-response meta-analysis to quantitatively assess the association between fluid intake and kidney stone risk. Based on a literature search of the PubMed, Embase, and Cochrane Library databases, 15 relevant studies (10 cohort and 5 case-control studies) were selected for inclusion in the meta-analysis with 9601 cases and 351,081 total participants.In the dose-response meta-analysis, we found that each 500 mL increase in water intake was associated with a significantly reduced risk of kidney stone formation (relative risk (RR) = 0.93; 95% CI: 0.87, 0.98; P < 0.01). Protective associations were also found for an increasing intake of tea (RR = 0.96; 95% CI: 0.93, 0.99; P = 0.02) and alcohol (RR = 0.80, 95% CI: 0.75, 0.85; P < 0.01). A borderline reverse association were observed on coffee intake and risk of kidney stone (RR = 0.88; 95% CI: 0.76, 1.00; P = 0.05). The risk of kidney stones was not significantly related to intake of juice (RR = 1.02, 95% CI: 0.95, 1.10; P = 0.64), soda (RR = 1.03; 95% CI: 0.90, 1.17; P = 0.65), or milk (RR = 0.96; 95% CI: 0.88, 1.03; P = 0.21). Subgroup analysis and sensitivity analyses showed inconsistent results on coffee, alcohol, and milk intake.Increased water intake is associated with a reduced risk of kidney stones; increased consumption of tea and alcohol may reduce kidney stone risk. An average daily water intake was recommended for kidney stone prevention.

Medical students do not adversely affect the quality of cardiopulmonary resuscitation for ED patients.

OBJECTIVES: To investigate the effect of medical student involvement on the quality of actual cardiopulmonary resuscitation (CPR). METHODS: A digital video-recording system was used to record and analyze CPR procedures for adult patients from March 2011 to September 2012. RESULTS: Twenty-six student-involved and 40 non-student-involved cases were studied. The chest compression rate in the student-involved group was significantly higher than that in the non-student-involved group (P < .001). The proportion of compressions at "above 110 cpm" was higher in the student-involved group (P = .021), whereas the proportion at "90-110 cpm" was lower in the student-involved group (P = .015). The ratio of hands-off time to total manual compression time was significantly lower in the student-involved group than in the non-student-involved group (P = .04). In contrast, the student-involved group delivered a higher ventilation rate compared with the non-student-involved group (P = .02). The observed time delay to first compression and first ventilation were very similar between the groups. There were no significant differences between the groups in either return of spontaneous circulation or time from survival to discharge. CONCLUSION: Student-involved resuscitation teams were able to perform good CPR, with higher compression rates and fewer interruptions. However, the supervision from medical staff is still needed to ensure appropriate chest compression and ventilation rate in student-involved actual CPR in the emergency department.