This article has been withdrawn by the authors. We discovered an error after this manuscript was published as a Paper in Press. Specifically, we learned that the structures of glycans presented for the PD-L1 peptide were drawn and labeled incorrectly. We wish to withdraw this article and submit a corrected version for review.

The histidine kinase Hik33 plays important roles in mediating cyanobacterial response to divergent types of abiotic stresses including cold, salt, high light (HL), and osmotic stresses. However, how these functions are regulated by Hik33 remains to be addressed. Using a hik33-deficient strain (hik33) of Synechocystis sp. PCC 6803 (Synechocystis) and quantitative proteomics, we found that Hik33 depletion induces differential protein expression highly similar to that induced by divergent types of stresses. This typically includes downregulation of proteins in photosynthesis and carbon assimilation that are necessary for cell propagation, and upregulation of heat shock proteins, chaperons, and proteases that are important for cell survival. This observation indicates that depletion of Hik33 alone mimics divergent types of abiotic stresses, and that Hik33 could be important for preventing abnormal stress response in the normal condition. Moreover, we found the majority of proteins of plasmid origin were significantly upregulated in hik33, though their biological significance remains to be addressed. Together, the systematically characterized Hik33-regulated cyanobacterial proteome, which is largely involved in stress responses, builds the molecular basis for Hik33 as a general regulator of stress responses.

Quantitative proteomics by mass spectrometry is widely used in biomarker research and basic biology research for investigation of phenotype level cellular events. Despite the wide application, the methodology for statistical analysis of differentially expressed proteins has not been unified. Various methods such as t-test, linear model and mixed effect models are used to define changes in proteomics experiments. However, none of these methods consider the specific structure of MS-data. Choices between methods, often originally developed for other types of data, are based on compromises between features such as statistical power, general applicability and user friendliness. Furthermore, whether to include proteins identified with one peptide in statistical analysis of differential protein expression varies between studies. Here we present DEqMS, a robust statistical method developed specifically for differential protein expression analysis in mass spectrometry data. In all datasets investigated there is a clear dependence of variance on the number of PSMs or peptides used for protein quantification. DEqMS takes this feature into account when assessing differential protein expression. This allows for a more accurate data-dependent estimation of protein variance and inclusion of single peptide identifications without increasing false discoveries. The method was tested in several datasets including E.coli proteome spike-in data, using both label-free and TMT-labelled quantification. In comparison to previous statistical methods used in quantitative proteomics, DEqMS showed consistently better accuracy in detecting altered protein levels compared to other statistical methods in both label-free and labelled quantitative proteomics data. DEqMS is available as an R package in Bioconductor.

Accumulation and propagation of hyperphosphorylated tau (p-tau) is a common neuropathological hallmark associated with neurodegeneration of Alzheimer's disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and related tauopathies. Extracellular vesicles, specifically exosomes, have recently been demonstrated to participate in mediating tau propagation in brain. Exosomes produced by human induced pluripotent stem cell (iPSC)-derived neurons expressing mutant Tau (mTau), containing the P301L and V337M Tau mutations of FTDP-17, possess the ability to propagate p-tau pathology after injection into mouse brain.  To gain an understanding of the mTau exosome cargo involved in tau pathogenesis, these pathogenic exosomes were analyzed by proteomics and bioinformatics. The data showed that mTau expression dysregulates the exosome proteome to result in (1) proteins uniquely present only in mTau, and not control exosomes, (2) the absence of proteins in mTau exosomes, uniquely present in control exosomes, and (3) shared proteins which were significantly up-regulated or down-regulated in mTau compared to control exosomes. Notably, mTau exosomes (not control exosomes) contain ANP32A (also known as I1PP2A), an endogenous inhibitor of the PP2A phosphatase which regulates the phosphorylation state of p-tau.  Several of the mTau exosome-specific proteins have been shown to participate in AD mechanisms involving lysosomes, inflammation, secretases, and related processes.  Furthermore, the mTau exosomes lacked a substantial portion of proteins present in control exosomes involved in pathways of localization, vesicle transport, and protein binding functions. The shared proteins present in both mTau and control exosomes represented exosome functions of vesicle-mediated transport, exocytosis, and secretion processes. These data illustrate mTau as a dynamic regulator of the biogenesis of exosomes to result in acquisition, deletion, and up- or down-regulation of protein cargo to result in pathogenic mTau exosomes capable of in vivo propagation of p-tau neuropathology in mouse brain. 

1 January 2007 ,

An ageing population in Europe makes the opinion of young people about their continent’s future even more important. Leading international figures, including the President of the United Nations General Assembly, have been expressing concern over the limited role of young people in shaping policy. With this in mind, The World Today and the National Association for Gifted and Talented Youth have given students the opportunity to share their vision through a competition on Europe: The Next Fifty Years. The three finalists have been invited to an International Economics Programme workshop at Chatham House next month.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are emerging as leading causes of liver disease worldwide and have been recognized as one of the major unmet medical needs of the 21st century. Our recent translational studies in mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine kinase (STK)25 as a protein that coats intrahepatocellular lipid droplets (LDs) and critically regulates liver lipid homeostasis and progression of NAFLD/NASH. Here, we studied the mechanism-of-action of STK25 in steatotic liver by relative quantification of the hepatic LD-associated phosphoproteome from high-fat diet-fed Stk25 knockout mice compared with their wild-type littermates. We observed a total of 131 proteins and 60 phosphoproteins that were differentially represented in STK25-deficient livers. Most notably, a number of proteins involved in peroxisomal function, ubiquitination-mediated proteolysis, and antioxidant defense were coordinately regulated in Stk25^–/– versus wild-type livers. We confirmed attenuated peroxisomal biogenesis and protection against oxidative and ER stress in STK25-deficient human liver cells, demonstrating the hepatocyte-autonomous manner of STK25’s action. In summary, our results suggest that regulation of peroxisomal function and metabolic stress response may be important molecular mechanisms by which STK25 controls the development and progression of NAFLD/NASH.

Angiopoietin-like protein 3 (ANGPTL3) inhibits lipid clearance and is a promising target for managing cardiovascular disease. Here we investigated the effects of a high-sugar (high-fructose) diet on circulating ANGPTL3 concentrations in rhesus macaques. Plasma ANGPTL3 concentrations increased ~30% to 40% after 1 and 3 months of a high-fructose diet (both P < 0.001 vs. baseline). During fructose-induced metabolic dysregulation, plasma ANGPTL3 concentrations were positively correlated with circulating indices of insulin resistance [assessed with fasting insulin and the homeostatic model assessment of insulin resistance (HOMA-IR)], hypertriglyceridemia, adiposity (assessed as leptin), and systemic inflammation [C-reactive peptide (CRP)] and negatively correlated with plasma levels of the insulin-sensitizing hormone adropin. Multiple regression analyses identified a strong association between circulating APOC3 and ANGPTL3 concentrations. Higher baseline plasma levels of both ANGPTL3 and APOC3 were associated with an increased risk for fructose-induced insulin resistance. Fish oil previously shown to prevent insulin resistance and hypertriglyceridemia in this model prevented increases of ANGPTL3 without affecting systemic inflammation (increased plasma CRP and interleukin-6 concentrations). ANGPTL3 RNAi lowered plasma concentrations of ANGPTL3, triglycerides (TGs), VLDL-C, APOC3, and APOE. These decreases were consistent with a reduced risk of atherosclerosis. In summary, dietary sugar-induced increases of circulating ANGPTL3 concentrations after metabolic dysregulation correlated positively with leptin levels, HOMA-IR, and dyslipidemia. Targeting ANGPTL3 expression with RNAi inhibited dyslipidemia by lowering plasma TGs, VLDL-C, APOC3, and APOE levels in rhesus macaques.

Atherosclerosis-related CVD causes nearly 20 million deaths annually. Most patients are treated after plaques develop, so therapies must regress existing lesions. Current therapies reduce plaque volume, but targeting all apoB-containing lipoproteins with intensive combinations that include alirocumab or evinacumab, monoclonal antibodies against cholesterol-regulating proprotein convertase subtilisin/kexin type 9 and angiopoietin-like protein 3, may provide more benefit. We investigated the effect of such lipid-lowering interventions on atherosclerosis in APOE*3-Leiden.CETP mice, a well-established model for hyperlipidemia. Mice were fed a Western-type diet for 13 weeks and thereafter matched into a baseline group (euthanized at 13 weeks) and five groups that received diet alone (control) or with treatment [atorvastatin; atorvastatin and alirocumab; atorvastatin and evinacumab; or atorvastatin, alirocumab, and evinacumab (triple therapy)] for 25 weeks. We measured effects on cholesterol levels, plaque composition and morphology, monocyte adherence, and macrophage proliferation. All interventions reduced plasma total cholesterol (37% with atorvastatin to 80% with triple treatment; all P < 0.001). Triple treatment decreased non-HDL-C to 1.0 mmol/l (91% difference from control; P < 0.001). Atorvastatin reduced atherosclerosis progression by 28% versus control (P < 0.001); double treatment completely blocked progression and diminished lesion severity. Triple treatment regressed lesion size versus baseline in the thoracic aorta by 50% and the aortic root by 36% (both P < 0.05 vs. baseline), decreased macrophage accumulation through reduced proliferation, and abated lesion severity. Thus, high-intensive cholesterol-lowering triple treatment targeting all apoB-containing lipoproteins regresses atherosclerotic lesion area and improves lesion composition in mice, making it a promising potential approach for treating atherosclerosis.

Loss of pancreatic β-cell mass and function as a result of sustained ER stress is a core step in the pathogenesis of diabetes mellitus type 2. The complex control of β-cells and insulin production involves hedgehog (Hh) signaling pathways as well as cholesterol-mediated effects. In fact, data from studies in humans and animal models suggest that HDL protects against the development of diabetes through inhibition of ER stress and β-cell apoptosis. We investigated the mechanism by which HDL inhibits ER stress and apoptosis induced by thapsigargin, a sarco/ER Ca²⁺-ATPase inhibitor, in β-cells of a rat insulinoma cell line, INS1e. We further explored effects on the Hh signaling receptor Smoothened (SMO) with pharmacologic agonists and inhibitors. Interference with sterol synthesis or efflux enhanced β-cell apoptosis and abrogated the anti-apoptotic activity of HDL. During ER stress, HDL facilitated the efflux of specific oxysterols, including 24-hydroxycholesterol (OHC). Supplementation of reconstituted HDL with 24-OHC enhanced and, in cells lacking ABCG1 or the 24-OHC synthesizing enzyme CYP46A1, restored the protective activity of HDL. Inhibition of SMO countered the beneficial effects of HDL and also LDL, and SMO agonists decreased β-cell apoptosis in the absence of ABCG1 or CYP46A1. The translocation of the SMO-activated transcription factor glioma-associated oncogene GLI-1 was inhibited by ER stress but restored by both HDL and 24-OHC. In conclusion, the protective effect of HDL to counter ER stress and β-cell death involves the transport, generation, and mobilization of oxysterols for activation of the Hh signaling receptor SMO

Fatty liver involves ectopic lipid accumulation and dysregulated hepatic oxidative metabolism, which can progress to a state of elevated inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH). The factors that control progression from simple steatosis to NASH are not fully known. Here, we tested the hypothesis that dietary vitamin E (VitE) supplementation would prevent NASH progression and associated metabolic alterations induced by a Western diet (WD). Hyperphagic melanocortin-4 receptor-deficient (MC4R^–/–) mice were fed chow, chow+VitE, WD, or WD+VitE starting at 8 or 20 weeks of age. All groups exhibited extensive hepatic steatosis by the end of the study (28 weeks of age). WD feeding exacerbated liver disease severity without inducing proportional changes in liver triglycerides. Eight weeks of WD accelerated liver pyruvate cycling, and 20 weeks of WD extensively upregulated liver glucose and oxidative metabolism assessed by ²H/¹³C flux analysis. VitE supplementation failed to reduce the histological features of NASH. Rather, WD+VitE increased the abundance and saturation of liver ceramides and accelerated metabolic flux dysregulation compared with 8 weeks of WD alone. In summary, VitE did not limit NASH pathogenesis in genetically obese mice, but instead increased some indicators of metabolic dysfunction.

Activation of microglia and astrocytes secondary to inflammatory processes contributes to the development and perpetuation of pain with a neuropathic phenotype. This pain state presents as a chronic debilitating condition and affects a large population of patients with conditions like rheumatoid arthritis and diabetes, or after surgery, trauma, or chemotherapy. Here, we review the regulation of lipid rafts in glial cells and the role they play as a key component of neuroinflammatory sensitization of central pain signaling pathways. In this context, we introduce the concept of an inflammaraft (i-raft), enlarged lipid rafts harboring activated receptors and adaptor molecules and serving as an organizing platform to initiate inflammatory signaling and the cellular response. Characteristics of the inflammaraft include increased relative abundance of lipid rafts in inflammatory cells, increased content of cholesterol per raft, and increased levels of inflammatory receptors, such as toll-like receptor (TLR)4, adaptor molecules, ion channels, and enzymes in lipid rafts. This inflammaraft motif serves an important role in the membrane assembly of protein complexes, for example, TLR4 dimerization. Operating within this framework, we demonstrate the involvement of inflammatory receptors, redox molecules, and ion channels in the inflammaraft formation and the regulation of cholesterol and sphingolipid metabolism in the inflammaraft maintenance and disruption. Strategies for targeting inflammarafts, without affecting the integrity of lipid rafts in noninflammatory cells, may lead to developing novel therapies for neuropathic pain states and other neuroinflammatory conditions.

Cholesterol/sphingolipid-rich membrane domains, known as lipid rafts or membrane rafts, play a critical role in the compartmentalization of signaling pathways. Physical segregation of proteins in lipid rafts may modulate the accessibility of proteins to regulatory or effector molecules. Thus, lipid rafts serve as sorting platforms and hubs for signal transduction proteins. Cancer cells contain higher levels of intracellular cholesterol and lipid rafts than their normal non-tumorigenic counterparts. Many signal transduction processes involved in cancer development (insulin-like growth factor system and phosphatidylinositol 3-kinase-AKT) and metastasis [cluster of differentiation (CD)44] are dependent on or modulated by lipid rafts. Additional proteins playing an important role in several malignant cancers (e.g., transmembrane glycoprotein mucin 1) are also being detected in association with lipid rafts, suggesting a major role of lipid rafts in tumor progression. Conversely, lipid rafts also serve as scaffolds for the recruitment and clustering of Fas/CD95 death receptors and downstream signaling molecules leading to cell death-promoting raft platforms. The partition of death receptors and downstream signaling molecules in aggregated lipid rafts has led to the formation of the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER, which leads to apoptosis amplification and can be pharmacologically modulated. These death-promoting rafts can be viewed as a linchpin from which apoptotic signals are launched. In this review, we discuss the involvement of lipid rafts in major signaling processes in cancer cells, including cell survival, cell death, and metastasis, and we consider the potential of lipid raft modulation as a promising target in cancer therapy.

When you run an SQL query using SAS/ACCESS Interface to ODBC under the following conditions, you might receive an error: You run SAS 9.4M5 (TS1M5) or SAS 9.4M6 (TS1M6)  i

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Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress–inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca2+ oscillations. As ER stress is known to reduce ER Ca2+ levels, we tested the hypothesis that the observed increase in Ca2+ oscillations occurred because of reduced ER Ca2+ levels and, in turn, increased store-operated Ca2+ entry. TM-induced cytosolic Ca2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca2+ depletion–activated Ca2+ current in mediating Ca2+-induced insulin secretion in response to ER stress.

Objective:

GPR40 is a G protein-coupled receptor regulating free fatty acid-induced insulin secretion. We have generated transgenic mice overexpressing the human GPR40 gene (hGPR40-Tg) under control of the mouse insulin II promoter and have used them to examine the role of GPR40 in the regulation of insulin secretion and glucose homeostasis.

To ensure fetal lipid supply, maternal blood triglyceride (TG) concentrations are robustly elevated during pregnancy. Interestingly, a lower increase in maternal blood TG concentrations has been observed in some obese mothers. We have shown that high-fat (HF) feeding during pregnancy significantly reduces maternal blood TG levels. Therefore, we performed this study to investigate if and how obesity alters maternal blood TG levels. Maternal obesity was established by prepregnant HF feeding (ppHF), which avoided the dietary effect during pregnancy. We found that maternal blood TG concentrations in ppHF dams were not only remarkably lower than control dams, but the TG peak occurred earlier during gestation. Hepatic TG production and intestinal TG absorption were unchanged in ppHF dams, but systemic lipoprotein lipase (LPL) activity was increased, suggesting that increased blood TG clearance contributes to the decreased blood TG concentrations in ppHF dams. Although significantly higher levels of UCP1 protein were observed in iBAT of ppHF dams, Ucp1 gene deletion did not restore blood TG concentrations in ppHF dams. Expression of the angiopoietin-like protein 4 (ANGPTL4), a potent endogenous LPL inhibitor, was significantly increased during pregnancy. However, the pregnancy-induced elevation of blood TG was almost abolished in Angptl4^-/- dams. Compared with control dams, Angptl4 mRNA levels were significantly lower in iBAT, gWAT and livers of ppHF dams. Importantly, ectopic overexpression of ANGPTL4 restored maternal blood TG concentrations in ppHF dams. Together, these results indicate that ANGPTL4 plays a vital role in increasing maternal blood TG concentrations during pregnancy. Obesity impairs the rise of maternal blood TG concentrations by reducing ANGPTL4 expression in mice.

Swi-independent 3a and 3b (Sin3a and Sin3b) are paralogous transcriptional coregulators that direct cellular differentiation, survival, and function. Here, we report that mouse Sin3a and Sin3b are co-produced in most pancreatic cells during embryogenesis but become much more enriched in endocrine cells in adults, implying continued essential roles in mature endocrine-cell function. Mice with loss of Sin3a in endocrine progenitors were normal during early postnatal stages but gradually developed diabetes before weaning. These physiological defects were preceded by the compromised survival, insulin-vesicle packaging, insulin secretion, and nutrient-induced Ca²⁺ influx of Sin3a-deficient β-cells. RNA-seq coupled with candidate chromatin-immunoprecipitation assays revealed several genes that could be directly regulated by Sin3a in β-cells, which modulate Ca²⁺/ion transport, cell survival, vesicle/membrane trafficking, glucose metabolism, and stress responses. Lastly, mice with loss of both Sin3a and Sin3b in multipotent embryonic pancreatic progenitors had significantly reduced islet-cell mass at birth, caused by decreased endocrine-progenitor production and increased β-cell death. These findings highlight the stage-specific requirements for the presumed "general" coregulators Sin3a and Sin3b in islet β-cells, with Sin3a being dispensable for differentiation but required for postnatal function and survival.

Insulin is first produced in pancreatic β-cells as the precursor prohormone proinsulin. Defective proinsulin processing has been implicated in the pathogenesis of both type 1 and type 2 diabetes. Though there is substantial evidence that mouse β-cells process proinsulin using prohormone convertase 1/3 (PC1/3) then prohormone convertase 2 (PC2), this finding has not been verified in human β-cells. Immunofluorescence with validated antibodies reveals that there was no detectable PC2 immunoreactivity in human β-cells and little PCSK2 mRNA by in situ hybridization. Similarly, rat β-cells were not immunoreactive for PC2. In all histological experiments, PC2 immunoreactivity in neighbouring α-cells acts as a positive control. In donors with type 2 diabetes, β-cells had elevated PC2 immunoreactivity, suggesting that aberrant PC2 expression may contribute to impaired proinsulin processing in β-cells of patients with diabetes. To support histological findings using a biochemical approach, human islets were used for pulse-chase experiments. Despite inhibition of PC2 function by temperature blockade, brefeldin-A, chloroquine, and multiple inhibitors that blocked production of mature glucagon from proglucagon, β-cells retained the ability to produce mature insulin. Conversely, suppression of PC1/3 blocked processing of proinsulin but not proglucagon. By demonstrating that healthy human β-cells process proinsulin by PC1/3 but not PC2 we suggest that there is a need to revise the longstanding theory of proinsulin processing.

Diabetes occurs due to a loss of functional β-cells, resulting from β-cell death and dysfunction. Lactogens protect rodent and human β-cells in vitro and in vivo against triggers of β-cell cytotoxicity relevant to diabetes, many of which converge onto a common pathway, endoplasmic reticulum (ER) stress. However, whether lactogens modulate the ER stress pathway is unknown. This study examines if lactogens can protect β-cells against ER stress and mitigate diabetes incidence in Akita mice, a rodent model of ER stress-induced diabetes, akin to neonatal diabetes in humans. We show that lactogens protect INS1 cells, primary rodent and human β-cells in vitro against two distinct ER stressors, tunicamycin and thapsigargin, through activation of the JAK2/STAT5 pathway. Lactogens mitigate expression of pro-apoptotic molecules in the ER stress pathway that are induced by chronic ER stress in INS1 cells and rodent islets. Transgenic expression of placental lactogen in β-cells of Akita mice drastically reduces the severe hyperglycemia, diabetes incidence, hypoinsulinemia, β-cell death, and loss of β-cell mass observed in Akita littermates. These are the first studies in any cell type demonstrating lactogens modulate the ER stress pathway, causing enhanced β-cell survival and reduced diabetes incidence in the face of chronic ER stress.

α-Klotho is a circulating factor with well-documented anti-aging properties; however, the central role of α-klotho in metabolism remains largely unexplored. The current study investigated the potential role of central α-klotho to modulate NPY/AgRP neurons, energy balance, and glucose homeostasis. Intracerebroventricular (ICV) administration of α-klotho suppressed food intake, improved glucose profiles, and reduced body weight in mouse models of Type I and II diabetes. Furthermore, central α-klotho inhibition via an anti-α-klotho antibody impaired glucose tolerance. Ex vivo patch clamp electrophysiology and immunohistochemical analysis revealed that α-klotho suppresses NPY/AgRP neuron activity, at least in part, by enhancing mIPSC’s. Experiments in hypothalamic GT1-7 cells observed α-klotho induces phosphorylation of AKT^ser473, ERK^{thr202/tyr204}, and FOXO1^ser256, as well as blunts AgRP gene transcription. Mechanistically, fibroblast growth factor 1 (FGFR1) inhibition abolished the downstream signaling of α-klotho, negated its ability to modulate NPY/AgRP neurons, and blunted its therapeutic effects. PI3 kinase inhibition also abolished α-klotho’s ability to suppress food intake and improve glucose clearance. These results indicate a prominent role of hypothalamic α-klotho/FGFR1/PI3K signaling in the modulation of NPY/AgRP neuron activity and maintenance of energy homeostasis, thus providing new insight into the pathophysiology of metabolic disease.

Hyperglycemia is a potent regulator of endogenous glucose production (EGP). Loss of this ‘glucose effectiveness’ is a major contributor to elevated plasma glucose concentrations in type 2 diabetes (T2D). ATP-sensitive potassium channels (K_ATP channels) in the central nervous system (CNS) have been shown to regulate EGP in humans and rodents. We examined the contribution of central K_ATP channels to glucose effectiveness. Under fixed hormonal conditions (‘pancreatic clamp’ studies), hyperglycemia suppressed EGP by ~50% in both non-diabetic humans and normal Sprague Dawley rats. By contrast, antagonism of K_ATP channels with glyburide significantly reduced the EGP-lowering effect of hyperglycemia in both humans and rats. Furthermore, the effects of glyburide on EGP and gluconeogenic enzymes in rats were abolished by intracerebroventricular (ICV) administration of the KATP channel agonist diazoxide. These findings indicate that about half of EGP suppression by hyperglycemia is mediated by central K_ATP channels. These central mechanisms may offer a novel therapeutic target for improving glycemic control in T2D.

Type 2 diabetes mellitus predicts outcome following acute myocardial infarction (AMI). Since underlying mechanics are incompletely understood, we investigated left ventricular (LV) and atrial (LA) pathophysiological changes and their prognostic implications using cardiovascular magnetic resonance (CMR). Consecutive patients (n=1147, n=265 diabetic; n=882 non-diabetic) underwent CMR 3 days after AMI. Analyses included LV ejection fraction (LVEF), global longitudinal, circumferential and radial strains (GLS, GCS and GRS), LA reservoir, conduit and booster pump strains, as well as infarct size, edema and microvascular obstruction. Predefined endpoints were major adverse cardiovascular events (MACE) within 12 months. Diabetic patients had impaired LA reservoir (19.8 vs. 21.2%, p<0.01) and conduit strains (7.6 vs. 9.0%, p<0.01) but not ventricular function or myocardial damage. They were at higher risk of MACE than non-diabetic patients (10.2% vs. 5.8%, p<0.01) with most MACE occurring in patients with LVEF≥35%. Whilst LVEF (p=0.045) and atrial reservoir strain (p=0.024) were independent predictors of MACE in non-diabetic patients, GLS was in diabetic patients (p=0.010). Considering patients with diabetes and LVEF≥35% (n=237), GLS and LA reservoir strain below median were significantly associated with MACE. In conclusion, in patients with diabetes, LA and LV longitudinal strain permit optimized risk assessment early after reperfused AMI with incremental prognostic value over and above LVEF.

Conceivably, upregulation of myo-inositol oxygenase (MIOX) is associated with altered cellular redox. Its promoter includes oxidant-response elements, and we also discovered binding sites for XBP-1, a transcription factor of ER stress response. Previous studies indicate that MIOX’s upregulation in acute tubular injury is mediated by oxidant and ER stress. Here, we investigated if hyperglycemia leads to accentuation of oxidant and ER stress, while boosting each other’s activities and thereby augmenting tubulo-interstitial injury/fibrosis. We generated MIOX-overexpressing transgenic (MIOX-TG) and -knockout (MIOX-KO) mice. A diabetic state was induced by streptozotocin administration. Also, MIOX-KO were crossbred with Ins2^Akita to generate Ins2^Akita/KO mice. MIOX-TG mice had worsening renal functions with kidneys having increased oxidant/ER stress, as reflected by DCF/DHE staining, perturbed NAD/NADH and GSH/GSSG ratios, increased NOX-4 expression, apoptosis and its executionary molecules, accentuation of TGF-β signaling, Smads and XBP-1 nuclear translocation, expression of GRP78 and XBP1 (ER stress markers) and accelerated tubulo-interstitial fibrosis. These changes were not seen in MIOX-KO mice. Interestingly, such changes were remarkably reduced in Ins2^Akita/KO mice, and likewise in vitro experiments with XBP1-siRNA. These findings suggest that MIOX expression accentuates while its deficiency shields kidneys from tubulo-interstitial injury by dampening oxidant and ER stress, which mutually enhance each other’s activity.

Obesity is a risk factor for type 2 diabetes (T2D), however not all obese individuals develop the disease. In this study, we aimed to investigate the cause of differential insulin secretion capacity of pancreatic islets from T2D and non-T2D (ND) especially obese donors (BMI ≥30 kg/m²). Islets from obese T2D donors had reduced insulin secretion, decreased β-cell exocytosis and higher expression of fatty acid translocase CD36. We tested the hypothesis that CD36 is a key molecule in the reduced insulin secretion capacity. Indeed, CD36 overexpression led to decreased insulin secretion, impaired exocytosis and reduced granule docking. This was accompanied with reduced expression of the exocytotic proteins, SNAP25, STXBP1 and VAMP2, likely because CD36 induced down-regulation of the IRS proteins, suppressed insulin signaling PI3K-AKT pathway and increased nuclear localization of the transcription factor FoxO1. CD36 antibody treatment of the human β-cell line, EndoC-βH1, increased IRS1 and exocytotic protein levels, improved granule docking and enhanced insulin secretion. Our results demonstrate that β-cells from obese T2D donors have dysfunctional exocytosis likely due to an abnormal lipid handling represented by differential CD36 expression. Hence, CD36 could be a key molecule to limit β-cell function in T2D associated with obesity.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neurotrophic factor widely expressed in mammalian tissues, and it exerts critical protective effects on neurons and other cell types in various disease models, such as those for diabetes. However, to date, the expression and roles of MANF in the cornea, with or without diabetic keratopathy (DK), remain unclear. Here, we demonstrate that MANF is abundantly expressed in normal corneal epithelial cells; however, MANF expression was significantly reduced in both unwounded and wounded corneal epithelium in streptozotocin-induced type 1 diabetic C57BL/6 mice. Recombinant human MANF significantly promoted normal and diabetic corneal epithelial wound healing and nerve regeneration. Furthermore, MANF inhibited hyperglycemia-induced endoplasmic reticulum (ER) stress and ER stress–mediated apoptosis. Attenuation of ER stress with 4-phenylbutyric acid (4-PBA) also ameliorated corneal epithelial closure and nerve regeneration. However, the beneficial effects of MANF and 4-PBA were abolished by an Akt inhibitor and Akt-specific small interfering RNA (siRNA). Finally, we reveal that the subconjunctival injection of MANF-specific siRNA prevents corneal epithelial wound healing and nerve regeneration. Our results provide important evidence that hyperglycemia-suppressed MANF expression may contribute to delayed corneal epithelial wound healing and impaired nerve regeneration by increasing ER stress, and MANF may be a useful therapeutic modality for treating DK.

Paraphrasing the Swiss physician and father of toxicology Paracelsus (1493–1541) on chemical agents used as therapeutics, "the dose makes the poison," it is now realized that this aptly applies to the calorigenic nutrients. The case here is the pancreatic islet β-cell presented with excessive levels of nutrients such as glucose, lipids, and amino acids. The short-term effects these nutrients exert on the β-cell are enhanced insulin biosynthesis and secretion and changes in glucose sensitivity. However, chronic fuel surfeit triggers additional compensatory and adaptive mechanisms by β-cells to cope with the increased insulin demand or to protect itself. When these mechanisms fail, toxicity due to the nutrient surplus ensues, leading to β-cell dysfunction, dedifferentiation, and apoptosis. The terms glucotoxicity, lipotoxicity, and glucolipotoxicity have been widely used, but there is some confusion as to what they mean precisely and which is most appropriate for a given situation. Here we address the gluco-, lipo-, and glucolipo-toxicities in β-cells by assessing the evidence both for and against each of them. We also discuss potential mechanisms and defend the view that many of the identified "toxic" effects of nutrient excess, which may also include amino acids, are in fact beneficial adaptive processes. In addition, candidate fuel-excess detoxification pathways are evaluated. Finally, we propose that a more general term should be used for the in vivo situation of overweight-associated type 2 diabetes reflecting both the adaptive and toxic processes to mixed calorigenic nutrients excess: "nutrient-induced metabolic stress" or, in brief, "nutri-stress."

In type 2 diabetes, β-cells endure various forms of cellular stress, including oxidative stress and endoplasmic reticulum stress, secondary to increased demand for insulin production and extracellular perturbations, including hyperglycemia. Chronic exposure to stress causes impaired insulin secretion, apoptosis, and loss of cell identity, and a combination of these processes leads to β-cell failure and severe hyperglycemia. Therefore, a better understanding of the molecular mechanisms underlying stress responses in β-cells promises to reveal new therapeutic opportunities for type 2 diabetes. In this perspective, we discuss posttranscriptional control of gene expression as a critical, but underappreciated, layer of regulation with broad importance during stress responses. Specifically, regulation of mRNA translation occurs pervasively during stress to activate gene expression programs; however, the convenience of RNA sequencing has caused translational regulation to be overlooked compared with transcriptional controls. We highlight the role of RNA binding proteins in shaping selective translational regulation during stress and the mechanisms underlying this level of regulation. A growing body of evidence indicates that RNA binding proteins control an array of processes in β-cells, including the synthesis and secretion of insulin. Therefore, systematic evaluations of translational regulation and the upstream factors shaping this level of regulation are critical areas of investigation to expand our understanding of β-cell failure in type 2 diabetes.

Prostate cancer (PCa) cells heavily rely on an active androgen receptor (AR) pathway for their survival. Enzalutamide (MDV3100) is a second-generation antiandrogenic drug that was approved by the Food and Drug Administration in 2012 to treat patients with castration-resistant prostate cancer (CRPC). However, emergence of resistance against this drug is inevitable, and it has been a major challenge to develop interventions that help manage enzalutamide-resistant CRPC. Erythropoietin-producing human hepatocellular (Eph) receptors are targeted by ephrin protein ligands and have a broad range of functions. Increasing evidence indicates that this signaling pathway plays an important role in tumorigenesis. Overexpression of EPH receptor B4 (EPHB4) has been observed in multiple types of cancer, being closely associated with proliferation, invasion, and metastasis of tumors. Here, using RNA-Seq analyses of clinical and preclinical samples, along with several biochemical and molecular methods, we report that enzalutamide-resistant PCa requires an active EPHB4 pathway that supports drug resistance of this tumor type. Using a small kinase inhibitor and RNAi-based gene silencing to disrupt EPHB4 activity, we found that these disruptions re-sensitize enzalutamide-resistant PCa to the drug both in vitro and in vivo. Mechanistically, we found that EPHB4 stimulates the AR by inducing proto-oncogene c-Myc (c-Myc) expression. Taken together, these results provide critical insight into the mechanism of enzalutamide resistance in PCa, potentially offering a therapeutic avenue for enhancing the efficacy of enzalutamide to better manage this common malignancy.

Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context–dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor–regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.

Accumulating evidence suggests that brown adipose tissue (BAT) is a potential therapeutic target for managing obesity and related diseases. PGAM family member 5, mitochondrial serine/threonine protein phosphatase (PGAM5), is a protein phosphatase that resides in the mitochondria and regulates many biological processes, including cell death, mitophagy, and immune responses. Because BAT is a mitochondria-rich tissue, we have hypothesized that PGAM5 has a physiological function in BAT. We previously reported that PGAM5-knockout (KO) mice are resistant to severe metabolic stress. Importantly, lipid accumulation is suppressed in PGAM5-KO BAT, even under unstressed conditions, raising the possibility that PGAM5 deficiency stimulates lipid consumption. However, the mechanism underlying this observation is undetermined. Here, using an array of biochemical approaches, including quantitative RT-PCR, immunoblotting, and oxygen consumption assays, we show that PGAM5 negatively regulates energy expenditure in brown adipocytes. We found that PGAM5-KO brown adipocytes have an enhanced oxygen consumption rate and increased expression of uncoupling protein 1 (UCP1), a protein that increases energy consumption in the mitochondria. Mechanistically, we found that PGAM5 phosphatase activity and intramembrane cleavage are required for suppression of UCP1 activity. Furthermore, utilizing a genome-wide siRNA screen in HeLa cells to search for regulators of PGAM5 cleavage, we identified a set of candidate genes, including phosphatidylserine decarboxylase (PISD), which catalyzes the formation of phosphatidylethanolamine at the mitochondrial membrane. Taken together, these results indicate that PGAM5 suppresses mitochondrial energy expenditure by down-regulating UCP1 expression in brown adipocytes and that its phosphatase activity and intramembrane cleavage are required for UCP1 suppression.

The developing nervous system is remarkably sensitive to environmental signals, including disruptive toxins, such as polybrominated diphenyl ethers (PBDEs). PBDEs are an environmentally pervasive class of brominated flame retardants whose neurodevelopmental toxicity mechanisms remain largely unclear. Using dissociated cortical neurons from embryonic Rattus norvegicus, we found here that chronic exposure to 6-OH–BDE-47, one of the most prevalent hydroxylated PBDE metabolites, suppresses both spontaneous and evoked neuronal electrical activity. On the basis of our previous work on mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) (MEK) biology and our observation that 6-OH–BDE-47 is structurally similar to kinase inhibitors, we hypothesized that certain hydroxylated PBDEs mediate neurotoxicity, at least in part, by impairing the MEK–ERK axis of MAPK signal transduction. We tested this hypothesis on three experimental platforms: 1) in silico, where modeling ligand–protein docking suggested that 6-OH–BDE-47 is a promiscuous ATP-competitive kinase inhibitor; 2) in vitro in dissociated neurons, where 6-OH–BDE-47 and another specific hydroxylated BDE metabolite similarly impaired phosphorylation of MEK/ERK1/2 and activity-induced transcription of a neuronal immediate early gene; and 3) in vivo in Drosophila melanogaster, where developmental exposures to 6-OH–BDE-47 and a MAPK inhibitor resulted in offspring displaying similarly increased frequency of mushroom-body β–lobe midline crossing, a metric of axonal guidance. Taken together, our results support that certain ortho-hydroxylated PBDE metabolites are promiscuous kinase inhibitors and can cause disruptions of critical neurodevelopmental processes, including neuronal electrical activity, pre-synaptic functions, MEK–ERK signaling, and axonal guidance.

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin–Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor–like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this post-translational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl acyltransferase, whose depletion affected barttin palmitoylation and ClC-K-barttin channel activation. We investigated the functional role of barttin palmitoylation in vivo in Zdhhc7−/− mice. Although palmitoylation of barttin in kidneys of Zdhhc7−/− animals was significantly decreased, it did not pathologically alter kidney structure and functions under physiological conditions. However, when Zdhhc7−/− mice were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) type IV. Of note, we also observed decreased palmitoylation of the disease-causing R8L barttin variant associated with human BS type IV. Our results indicate that dysregulated DHHC7-mediated barttin palmitoylation appears to play an important role in chloride channel dysfunction in certain BS variants, suggesting that targeting DHHC7 activity may offer a potential therapeutic strategy for reducing hypertension.

Optic atrophy 1 (OPA1) is a dynamin protein that mediates mitochondrial fusion at the inner membrane. OPA1 is also necessary for maintaining the cristae and thus essential for supporting cellular energetics. OPA1 exists as membrane-anchored long form (L-OPA1) and short form (S-OPA1) that lacks the transmembrane region and is generated by cleavage of L-OPA1. Mitochondrial dysfunction and cellular stresses activate the inner membrane–associated zinc metallopeptidase OMA1 that cleaves L-OPA1, causing S-OPA1 accumulation. The prevailing notion has been that L-OPA1 is the functional form, whereas S-OPA1 is an inactive cleavage product in mammals, and that stress-induced OPA1 cleavage causes mitochondrial fragmentation and sensitizes cells to death. However, S-OPA1 contains all functional domains of dynamin proteins, suggesting that it has a physiological role. Indeed, we recently demonstrated that S-OPA1 can maintain cristae and energetics through its GTPase activity, despite lacking fusion activity. Here, applying oxidant insult that induces OPA1 cleavage, we show that cells unable to generate S-OPA1 are more sensitive to this stress under obligatory respiratory conditions, leading to necrotic death. These findings indicate that L-OPA1 and S-OPA1 differ in maintaining mitochondrial function. Mechanistically, we found that cells that exclusively express L-OPA1 generate more superoxide and are more sensitive to Ca2+-induced mitochondrial permeability transition, suggesting that S-OPA1, and not L-OPA1, protects against cellular stress. Importantly, silencing of OMA1 expression increased oxidant-induced cell death, indicating that stress-induced OPA1 cleavage supports cell survival. Our findings suggest that S-OPA1 generation by OPA1 cleavage is a survival mechanism in stressed cells.

Endorepellin, the C-terminal fragment of the heparan sulfate proteoglycan perlecan, influences various signaling pathways in endothelial cells by binding to VEGFR2. In this study, we discovered that soluble endorepellin activates the canonical stress signaling pathway consisting of PERK, eIF2α, ATF4, and GADD45α. Specifically, endorepellin evoked transient activation of VEGFR2, which, in turn, phosphorylated PERK at Thr980. Subsequently, PERK phosphorylated eIF2α at Ser51, upregulating its downstream effector proteins ATF4 and GADD45α. RNAi-mediated knockdown of PERK or eIF2α abrogated the endorepellin-mediated up-regulation of GADD45α, the ultimate effector protein of this stress signaling cascade. To functionally validate these findings, we utilized an ex vivo model of angiogenesis. Exposure of the aortic rings embedded in 3D fibrillar collagen to recombinant endorepellin for 2–4 h activated PERK and induced GADD45α vis à vis vehicle-treated counterparts. Similar effects were obtained with the established cellular stress inducer tunicamycin. Notably, chronic exposure of aortic rings to endorepellin for 7–9 days markedly suppressed vessel sprouting, an angiostatic effect that was rescued by blocking PERK kinase activity. Our findings unravel a mechanism by which an extracellular matrix protein evokes stress signaling in endothelial cells, which leads to angiostasis.