Under UNIX, the process of updating a lookup table in SAS Business Rules Manager does not work properly. The problem occurs when you perform these steps:  Open a lookup table. Cl

SAS Visual Data Builder might not enable you to create multiple time-event triggers. The + button to add another trigger is not available to select, as shown in the following display: imgalt="" src="{fusion_658

When you attempt to save a report distribution in SAS Visual Analytics Designer, you might see the error shown in the following display:  imgalt="" src="{fusion_65868_1_distributionerror.png}" />

Baseball season is now on the clock and the Rays are looking to get back to the postseason for the first time since 2014. That quest will begin next week as Spring Training is finally upon us. Here's a primer to get you informed on all you need to know this spring.

Over the past couple seasons, the Rays have preached patience as the organization provided time for the top prospects to make it up to the Majors. Now, the focus has been primarily in remaining flexible and keeping positions open for the young talent arriving from the Minors.

Here's an early look at how the Rays' 25-man roster could shape up on Opening Day.

All 30 Major League teams will wear special "MLB 150" patches on their uniforms for the entire 2019 season in honor of the 150th anniversary of the 1869 Cincinnati Red Stockings, the first openly all-salaried professional baseball team.

Brendan McKay is still going to be a two-way player, but the Rays are planning to narrow his focus on the hitting side by keeping him to designated hitter-only duties, a source confirmed to MLB.com's Juan Toribio on Monday.

After winning the 2018 American League Cy Young Award, Blake Snell said he doesn't feel "any pressure" in '19.

When Tyler Glasnow heard the news that the Rays were signing Charlie Morton, he couldn't help but get even more excited for the 2019 season.

The 30 prospects below all are getting very long looks this spring with an eye toward breaking camp with the parent club. Even if they start the year in the Minors, they all should get the chance to contribute at some point in the very near future.

Colin Poche's fastball won't light up the radar gun, but it proved to be one of the most effective pitches in the Minors last season.

Who do the Rays have in the pipeline? Get scouting reports, video, stats, projected ETAs and more for Tampa Bay's Top 30 Prospects on MLB Pipeline's Prospect Watch.

The next five weeks will see lots of shuffling on Major League rosters. Here are the most intriguing positional battles on each of the 30 MLB clubs.

While most of the attention will fall on the players who are expected to make the Opening Day roster, manager Kevin Cash and the rest of the organization will be keeping a close eye on some of the players that could find their way to the big leagues at some point within the next year.

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5–ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5–ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5–ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte–EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation.

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.

Diabetic retinopathy (DR) is diagnosed clinically by directly viewing retinal vascular changes during ophthalmoscopy or through fundus photographs. However, electroretinography (ERG) studies in humans and rodents have revealed that retinal dysfunction is demonstrable prior to the development of visible vascular defects. Specifically, delays in dark-adapted ERG oscillatory potential (OP) implicit times in response to dim flash stimuli (<-1.8 log cd·s/m²) occur prior to clinically-recognized diabetic retinopathy. Animal studies suggest that retinal dopamine deficiency underlies these early functional deficits. Here, we randomized persons with diabetes, without clinically detectable retinopathy, to treatment with either low or high dose Sinemet (levodopa plus carbidopa) for 2 weeks and compared their ERG findings with those of control (no DM) subjects. We assessed dim flash stimulated OP delays using a novel hand-held ERG system (RETeval) at baseline, 2 and 4 weeks. RETeval recordings identified significant OP implicit-time delays in persons with diabetes without retinopathy compared to age-matched controls (p<0.001). After two weeks of Sinemet treatment, OP implicit times were restored to control values, and these improvements persisted even after a two-week washout. We conclude that detection of dim flash OP delays could provide early detection of DR, and that Sinemet treatment may reverse retinal dysfunction.

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.

Clinical studies have shown a link between hyperuricemia (HU) and diabetes, while the exact effect of soluble serum urate on glucose metabolism remains elusive. This study aims to characterize the glucose metabolic phenotypes and investigate the underlying molecular mechanisms using a novel spontaneous HU mouse model in which the Uricase (Uox) gene is absent. In an attempt to study the role of HU in glycometabolism, we implemented external stimulation on Uox-knockout (KO) and wild-type (WT) males with a high-fat diet (HFD) and/or injections of multiple low-dose streptozotocin (MLD-STZ) to provoke the potential role of urate. Notably, while Uox-KO mice developed glucose intolerance in the basal condition, no mice spontaneously developed diabetes, even with aging. HFD-fed Uox-KO mice manifested similar insulin sensitivity compared with WT controls. HU augmented the existing glycometabolism abnormality induced by MLD-STZ and eventually led to diabetes, as evidenced by the increased random glucose. Reduced β-cell masses and increased terminal deoxynucleotidyl TUNEL-positive β-cells suggested that HU-mediated diabetes was cell death dependent. However, urate-lowering treatment (ULT) cannot ameliorate the diabetes incidence or reverse β-cell apoptosis with significance. ULT displayed a significant therapeutic effect of HU-crystal– associated kidney injury and tubulointerstitial damage in diabetes. Moreover, we present transcriptomic analysis of isolated islets, using Uox-KO versus WT mice and streptozotocin-induced diabetic WT (STZ-WT) versus diabetic Uox-KO (STZ-KO) mice. Shared differentially expressed genes of HU primacy revealed Stk17β is a possible target gene in HU-related β-cell death. Together, this study suggests that HU accelerates but does not cause diabetes by inhibiting islet β-cell survival.

Chronic low-grade inflammation plays a central role in the pathophysiology of gestational diabetes mellitus (GDM). In order to investigate the ability of different inflammatory blood cell parameters in predicting the development of GDM and pregnancy outcomes, 258 women with GDM and 1154 women without were included in this retrospective study. First-trimester neutrophil count outperformed white blood cell (WBC) count, and neutrophil-to-lymphocyte ratio (NLR) in the predictability for GDM. Subjects were grouped based on tertiles of neutrophil count during their first-trimester pregnancy. The results showed that as the neutrophil count increased, there was a step-wise increase in GDM incidence, as well as glucose and glycosylated hemoglobin (HbA1c) level, Homeostasis Model Assessment for Insulin Resistance (HOMA-IR), macrosomia incidence and newborn weight. Neutrophil count was positively associated with pre-pregnancy Body Mass Index (BMI), HOMA-IR and newborn weight. Additionally, neutrophil count was an independent risk factor for the development of GDM, regardless of the history of GDM. Spline regression showed that there was a significant linear association between GDM incidence and continuous neutrophil count when it exceeded 5.0 x 10⁹/L. This work suggested that first-trimester neutrophil count is closely associated with the development of GDM and adverse pregnancy outcomes.

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.

The brain mechanisms underlying the association of hyperglycemia with depressive symptoms are unknown. We hypothesized that disrupted glutamate metabolism in pregenual anterior cingulate cortex (ACC) in type 1 diabetes (T1D) without depression affects emotional processing. Using proton magnetic resonance spectroscopy (MRS), we measured glutamate concentrations in ACC and occipital cortex (OCC) in 13 T1D without major depression (HbA1c=7.1±0.7% [54±7mmol/mol]) and 11 healthy non-diabetic controls (HbA1c=5.5±0.2% [37±3mmol/mol]) during fasting euglycemia (EU) followed by a 60-minute +5.5mmol/l hyperglycemic clamp (HG). Intrinsic neuronal activity was assessed using resting-state blood oxygen level dependent functional MRI to measure the fractional amplitude of low frequency fluctuations in slow-band 4 (fALFF4). Emotional processing and depressive symptoms were assessed using emotional tasks (Emotional-Stroop, Self-Referent-Encoding-Task SRET) and clinical ratings (HAM-D, SCL-90-R), respectively. During HG, ACC glutamate increased (1.2mmol/kg, +10%, p=0.014) while ACC fALFF4 was unchanged (-0.007, -2%, p=0.449) in T1D; in contrast, glutamate was unchanged (-0.2mmol/kg, -2%, p=0.578) while fALFF4 decreased (-0.05, -13%, p=0.002) in controls. OCC glutamate and fALFF4 were unchanged in both groups. T1D had longer SRET negative-word response-times (p=0.017) and higher depression-rating scores (HAM-D p=0.020; SCL-90-R-depression p=0.008). Higher glutamate change tended to associate with longer Emotional-Stroop response-times in T1D only. Brain glutamate must be tightly controlled during hyperglycemia due to the risk for neurotoxicity with excessive levels. Results suggest that ACC glutamate control mechanisms are disrupted in T1D, which affects glutamatergic neurotransmission related to emotional or cognitive processing. Increased prefrontal glutamate during acute hyperglycemic episodes could explain our previous findings of associations between chronic hyperglycemia, cortical thinning and depressive symptoms in T1D.

Obesity has recently become a prevalent health threat worldwide. Although emerging evidence has suggested a strong link between the pentose phosphate pathway (PPP) and obesity, the role of transketolase (TKT), an enzyme in the non-oxidative branch of the PPP which connects PPP and glycolysis, remains obscure in adipose tissues. In this study, we specifically delete TKT in mouse adipocytes and find no obvious phenotype upon normal diet feeding. However, adipocyte TKT abrogation attenuates high fat diet (HFD)-induced obesity, reduces hepatic steatosis, improves glucose tolerance, alleviates insulin resistance and increases energy expenditure. Mechanistically, TKT deficiency accumulates non-oxidative PPP metabolites, decreases glycolysis and pyruvate input into the mitochondria, leading to increased lipolytic enzyme gene expression and enhanced lipolysis, fatty acid oxidation and mitochondrial respiration. Therefore, our data not only identify a novel role of TKT in regulating lipolysis and obesity, but also suggest limiting glucose-derived carbon into the mitochondria induces lipid catabolism and energy expenditure.

Hypo-vascularised diabetic non-healing wounds are due to reduced number and impaired physiology of endogenous endothelial progenitor cell (EPC) population that, limits their recruitment and mobilization at the wound site. To enrich the EPC repertoire from non-endothelial precursors, abundantly available mesenchymal stromal cells (MSCs) were reprogrammed into induced-endothelial cells (iECs). We identified cell signaling molecular targets by meta-analysis of microarray datasets. BMP-2 induction leads to the expression of inhibitory Smad 6/7-dependent negative transcriptional regulation of ID1, rendering the latter's reduced binding to TWIST1 during transdifferentiation of WJ-MSC into iEC. TWIST1, in turn, regulates endothelial genes transcription, positively of pro-angiogenic-KDR and negatively, in part, of anti-angiogenic-SFRP4. Twist1 reprogramming enhanced the endothelial lineage commitment of WJ-MSC, increased the vasculogenic potential of reprogrammed EC (rEC). Transplantation of stable TWIST1-rECs into full-thickness type 1 and 2 diabetic-splinted wound healing murine model enhanced the microcirculatory blood flow and accelerated the wound tissue regeneration. An increased or decreased co-localization of GFP with KDR/SFRP4 and CD31 in the regenerated diabetic wound bed with TWIST1 overexpression or silencing (piLenti-TWIST1-shRNA-GFP), respectively further confirmed improved neovascularization. This study depicted the reprogramming of WJ-MSCs into rECs using unique transcription factors, TWIST1 for an efficacious cell transplantation therapy to induce neovascularization–mediated diabetic wound tissue regeneration.

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.

Mobilization of hematopoietic stem/progenitor cells (HSPCs) from the bone marrow (BM) is impaired in diabetes. Excess oncostatin M (OSM) produced by M1 macrophages in the diabetic BM signals through p66Shc to induce Cxcl12 in stromal cells and retain HSPCs. BM adipocytes are another source of CXCL12 that blunts mobilization. We tested a strategy of pharmacologic macrophage reprogramming to rescue HSPC mobilization. In vitro, PPAR- activation with pioglitazone switched macrophages from M1 to M2, reduced Osm expression, and prevented transcellular induction of Cxcl12. In diabetic mice, pioglitazone treatment downregulated Osm, p66Shc and Cxcl12 in the hematopoietic BM, restored the effects of granulocyte-colony stimulation factor (G-CSF), and partially rescued HSPC mobilization, but it increased BM adipocytes. Osm deletion recapitulated the effects of pioglitazone on adipogenesis, which was p66Shc-independent, and double knockout of Osm and p66Shc completely rescued HSPC mobilization. In the absence of OSM, BM adipocytes produced less CXCL12, being arguably devoid of HSPC-retaining activity, whereas pioglitazone failed to downregulate Cxcl12 in BM adipocytes. In diabetic patients under pioglitazone therapy, HSPC mobilization after G-CSF was partially rescued. In summary, pioglitazone reprogrammed BM macrophages and suppressed OSM signaling, but sustained Cxcl12 expression by BM adipocytes could limit full recovery of HSPC mobilization.

Previous studies show that 12-week of high-fat diet (HFD) consumption caused not only prediabetes, but also cognitive decline and brain pathologies. Recently, necrostatin-1 (nec-1), a necroptosis inhibitor, showed beneficial effects in brain against stroke. However, the comparative effects of nec-1 and metformin on cognition and brain pathologies in prediabetes have not been investigated. We hypothesized that nec-1 and metformin equally attenuated cognitive decline and brain pathologies in prediabetic rats. Rats (n=32) were fed with either normal diet (ND) or high-fat diet (HFD) for 20 weeks. At week 13, ND-fed rats were given a vehicle (n=8) and HFD-fed rats were randomly assigned into 3 subgroups (n=8/subgroup) with vehicle, nec-1 or metformin for 8 weeks. Metabolic parameters, cognitive function, brain insulin receptor function, synaptic plasticity, dendritic spine density, microglial morphology, brain mitochondrial function, Alzheimer’s protein, and cell death were determined. HFD-fed rats exhibited prediabetes, cognitive decline, and brain pathologies. Nec-1 and metformin equally improved cognitive function, synaptic plasticity, dendritic spine density, microglial morphology, brain mitochondrial function, reduced hyperphosphorylated-tau and necroptosis in HFD-fed rats. Interestingly metformin, but not nec-1, improved brain insulin sensitivity in those rats. In conclusion, necroptosis inhibition directly improved cognition in prediabetic rats without alteration in insulin sensitivity.

Monogenic forms of obesity have been identified in ≤10% of severely obese European patients. However, the overall spectrum of deleterious variants (point mutations and structural variants) responsible for childhood severe obesity remains elusive. In this study, we genetically screened 225 severely obese children from consanguineous Pakistani families through a combination of techniques including an in-house developed augmented whole-exome sequencing (CoDE-seq) enabling simultaneous detection of whole exome copy number variations (CNVs) and of point mutations in coding regions. We identified 110 probands (49%) carrying 55 different pathogenic point mutations and CNVs in 13 genes/loci responsible for non-syndromic and syndromic monofactorial obesity. CoDE-seq also identified 28 rare or novel CNVs associated with intellectual disability in 22 additional obese subjects (10%). Additionally, we highlight variants in candidate genes for obesity warranting further investigation. Altogether, 59% of the studied cohort are likely to have a discrete genetic cause with 13% of these due to CNVs demonstrating a remarkably higher prevalence of monofactorial obesity than hitherto reported and a plausible over lapping of obesity and intellectual disabilities in several cases. Finally, inbred populations with high prevalence of obesity, provide a unique genetically enriched material in quest of new genes/variants influencing energy balance.

Infants born to mothers with obesity have a greater risk for childhood obesity and metabolic diseases; however, the underlying biological mechanisms remain poorly understood. We used a Japanese macaque model to investigate whether maternal obesity combined with a western-style diet (WSD) impairs offspring muscle insulin action. Adult females were fed a control or WSD prior to and during pregnancy through lactation, and offspring subsequently weaned to a control or WSD. Muscle glucose uptake and signaling were measured ex vivo in fetal (n=5-8/group) and juvenile offspring (n=8/group). In vivo signaling was evaluated after an insulin bolus just prior to weaning (n=4-5/group). Maternal WSD reduced insulin-stimulated glucose uptake and impaired insulin signaling at the level of Akt phosphorylation in fetal muscle. In juvenile offspring, insulin-stimulated glucose uptake was similarly reduced by both maternal and post-weaning WSD and corresponded to modest reductions in insulin-stimulated Akt phosphorylation relative to controls. We conclude that maternal WSD leads to a persistent decrease in offspring muscle insulin-stimulated glucose uptake even in the absence of increased offspring adiposity or markers of systemic insulin resistance. Switching offspring to a healthy diet did not reverse the effects of maternal WSD on muscle insulin action suggesting earlier interventions may be warranted.

Nonalcoholic steatohepatitis has emerged as a major cause of liver diseases with no effective therapies. Here, we evaluate the efficacies and pharmacokinetics of B1344, a long-acting PEGylated FGF21 analog, in a nongenetically modified nonhuman primate species that underwent liver biopsy, and demonstrate the potential for efficacies in humans. B1344 is sufficient to selectively activate signaling from the βKlotho/FGFR1c receptor complex. In cynomolgus monkeys with nonalcoholic fatty liver disease, administration of B1344 via subcutaneous injection for eleven weeks caused a profound reduction of hepatic steatosis, inflammation and fibrosis, and amelioration of liver injury and hepatocyte death as evidenced by liver biopsy and biochemical analysis. Moreover, improvement of metabolic parameters was observed in the monkey, including reduction of body weight and improvement of lipid profiles and glycemic control. To determine the role of B1344 in the progression of murine NAFLD independent of obesity, administration of B1344 were performed in mice fed with methionine and choline deficiency diet. Consistently, B1344 administration prevented the mice from lipotoxicity damage and nonalcoholic steatohepatitis at a dose-dependent manner. These results provide preclinical validation for an innovative therapeutics to NAFLD, and support further clinical testing of B1344 for treating nonalcoholic steatohepatitis and other metabolic diseases in humans.

A failure in self-tolerance leads to autoimmune destruction of pancreatic β-cells and type 1 diabetes (T1D). Low molecular weight dextran sulfate (DS) is a sulfated semi-synthetic polysaccharide with demonstrated cytoprotective and immunomodulatory properties in vitro. However, whether DS can protect pancreatic β-cells, reduce autoimmunity and ameliorate T1D is unknown. Here we report that DS, but not dextran, protects human β-cells against cytokine-mediated cytotoxicity in vitro. DS also protects mitochondrial function and glucose-stimulated insulin secretion and reduces chemokine expression in human islets in a pro-inflammatory environment. Interestingly, daily treatment with DS significantly reduces diabetes incidence in pre-diabetic non-obese diabetic (NOD) mice, and most importantly, reverses diabetes in early-onset diabetic NOD mice. DS decreases β-cell death, enhances islet heparan sulfate (HS)/heparan sulfate proteoglycan (HSPG) expression and preserves β-cell mass and plasma insulin in these mice. DS administration also increases the expression of the inhibitory co-stimulatory molecule programmed death-1 (PD-1) in T-cells, reduces interferon-+ CD4+ and CD8+ T-cells and enhances the number of FoxP3+ cells. Collectively, these studies demonstrate that the action of one single molecule, DS, on β-cell protection, extracellular matrix preservation and immunomodulation can reverse diabetes in NOD mice highlighting its therapeutic potential for the treatment of T1D.

Islet transplantation is an emerging therapy for type 1 diabetes (T1D) and hypoglycaemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study we established a diabetic-humanized NOD-scidIL2R^null(NSG) mouse model of T cell mediated human islet-allograft rejection and developed a therapeutic regimen of low-dose recombinant human interleukin2(IL-2) combined with low-dose rapamycin to prolong graft survival. NSG-mice that had received renal-subcapsular human islet-allografts and were transfused with 1x10⁷ of human-spleen-mononuclear-cells (hSPMCs), reconstituted human CD45⁺ cells that were predominantly CD3⁺ T cells and rejected their grafts with a median survival time of 27 days. IL-2 alone (0.3x10⁶ IU/m² or 1x10⁶ IU/m²), or rapamycin alone (0.5-1mg/kg) for 3 weeks did not prolong survival. However, the combination of rapamycin with IL-2 for 3 weeks significantly prolonged human islet-allograft survival. Graft survival was associated with expansion of CD4⁺CD25⁺FOXP3⁺ Tregs and enhanced TGF-β production by CD4⁺ T cells. CD8⁺ T cells showed reduced IFN- production and reduced expression of perforin-1. The combination of IL-2 and rapamycin has the potential to inhibit human islet-allograft rejection by expanding CD4⁺FOXP3⁺ Tregs in vivo and supressing effector cell function, and could be the basis of effective tolerance-based regimens.

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.

The increasing prevalence of type 2 diabetes poses a major challenge to societies worldwide. Blood-based factors like serum proteins are in contact with every organ in the body to mediate global homeostasis and may thus directly regulate complex processes such as aging and the development of common chronic diseases. We applied a data-driven proteomics approach, measuring serum levels of 4,137 proteins in 5,438 elderly Icelanders and identified 536 proteins associated with prevalent and/or incident type 2 diabetes. We validated a subset of the observed associations in an independent case-control study of type 2 diabetes. These protein associations provide novel biological insights into the molecular mechanisms that are dysregulated prior to and following the onset of type 2 diabetes and can be detected in serum. A bi-directional two-sample Mendelian randomization analysis indicated that serum changes of at least 23 proteins are downstream of the disease or its genetic liability, while 15 proteins were supported as having a causal role in type 2 diabetes.

Diabetes is now a pandemic disease. Moreover, a large number of people with prediabetes are at risk for developing frank diabetes worldwide. Both type 1 and type 2 diabetes increase the risk of atherosclerotic cardiovascular disease (CVD). Even with statin treatment to lower LDL cholesterol, patients with diabetes have a high residual CVD risk. Factors mediating the residual risk are incompletely characterized. An attractive hypothesis is that remnant lipoprotein particles (RLPs), derived by lipolysis from VLDL and chylomicrons, contribute to this residual risk. RLPs constitute a heterogeneous population of lipoprotein particles, varying markedly in size and composition. Although a universally accepted definition is lacking, for the purpose of this review we define RLPs as postlipolytic partially triglyceride-depleted particles derived from chylomicrons and VLDL that are relatively enriched in cholesteryl esters and apolipoprotein (apo)E. RLPs derived from chylomicrons contain apoB48, while those derived from VLDL contain apoB100. Clarity as to the role of RLPs in CVD risk is hampered by lack of a widely accepted definition and a paucity of adequate methods for their accurate and precise quantification. New specific methods for RLP quantification would greatly improve our understanding of their biology and role in promoting atherosclerosis in diabetes and other disorders.

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.

The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100–184 or 100–200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.

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.

Kindlins are focal adhesion proteins that regulate integrin activation and outside-in signaling. The kindlin family consists of three members, kindlin-1, -2, and -3. Kindlin-2 is widely expressed in multiple cell types, except those from the hematopoietic lineage. A previous study has reported that the Drosophila Fit1 protein (an ortholog of kindlin-2) prevents abnormal spindle assembly; however, the mechanism remains unknown. Here, we show that kindlin-2 maintains spindle integrity in mitotic human cells. The human neuroblastoma SH-SY5Y cell line expresses only kindlin-2, and we found that when SH-SY5Y cells are depleted of kindlin-2, they exhibit pronounced spindle abnormalities and delayed mitosis. Of note, acetylation of α-tubulin, which maintains microtubule flexibility and stability, was diminished in the kindlin-2–depleted cells. Mechanistically, we found that kindlin-2 maintains α-tubulin acetylation by inhibiting the microtubule-associated deacetylase histone deacetylase 6 (HDAC6) via a signaling pathway involving AKT Ser/Thr kinase (AKT)/glycogen synthase kinase 3β (GSK3β) or paxillin. We also provide evidence that prolonged hypoxia down-regulates kindlin-2 expression, leading to spindle abnormalities not only in the SH-SY5Y cell line, but also cell lines derived from colon and breast tissues. The findings of our study highlight that kindlin-2 regulates mitotic spindle assembly and that this process is perturbed in cancer cells in a hypoxic environment.