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When Yandy Diaz arrived at Rays camp on Sunday, he quickly established himself as the most muscular player inside the clubhouse. During Spring Training, his focus will be to establish himself as an everyday player.

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+]i), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+]i. Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.

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.

T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1–IS2 and IS3–IS4 loops of domain I and a cluster of extracellular cysteines in the IS1–IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species–producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1–IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1–IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.

Pathogenic bacteria of the genera Mycobacterium and Corynebacterium cause severe human diseases such as tuberculosis (Mycobacterium tuberculosis) and diphtheria (Corynebacterium diphtheriae). The cells of these species are surrounded by protective cell walls rich in long-chain mycolic acids. These fatty acids are conjugated to the disaccharide trehalose on the cytoplasmic side of the bacterial cell membrane. They are then transported across the membrane to the periplasm where they act as donors for other reactions. We have previously shown that transient acetylation of the glycolipid trehalose monohydroxycorynomycolate (hTMCM) enables its efficient transport to the periplasm in Corynebacterium glutamicum and that acetylation is mediated by the membrane protein TmaT. Here, we show that a putative methyltransferase, encoded at the same genetic locus as TmaT, is also required for optimal hTMCM transport. Deletion of the C. glutamicum gene NCgl2764 (Rv0224c in M. tuberculosis) abolished acetyltrehalose monocorynomycolate (AcTMCM) synthesis, leading to accumulation of hTMCM in the inner membrane and delaying its conversion to trehalose dihydroxycorynomycolate (h2TDCM). Complementation with NCgl2764 normalized turnover of hTMCM to h2TDCM. In contrast, complementation with NCgl2764 derivatives mutated at residues essential for methyltransferase activity failed to rectify the defect, suggesting that NCgl2764/Rv0224c encodes a methyltransferase, designated here as MtrP. Comprehensive analyses of the individual mtrP and tmaT mutants and of a double mutant revealed strikingly similar changes across several lipid classes compared with WT bacteria. These findings indicate that both MtrP and TmaT have nonredundant roles in regulating AcTMCM synthesis, revealing additional complexity in the regulation of trehalose mycolate transport in the Corynebacterineae.

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.

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D). We aimed to identify the peripheral blood DNA methylation signature of hepatic fat. We conducted epigenome-wide association studies of hepatic fat in 3,400 European ancestry (EA) participants and in 401 Hispanic ancestry and 724 African ancestry participants from four population-based cohort studies. Hepatic fat was measured using computed tomography or ultrasound imaging and DNA methylation was assessed at >400,000 cytosine-guanine dinucleotides (CpGs) in whole blood or CD14+ monocytes using a commercial array. We identified 22 CpGs associated with hepatic fat in EA participants at a false discovery rate <0.05 (corresponding P = 6.9 x 10^–6) with replication at Bonferroni-corrected P < 8.6 x 10^–4. Mendelian randomization analyses supported the association of hypomethylation of cg08309687 (LINC00649) with NAFLD (P = 2.5 x 10^–4). Hypomethylation of the same CpG was also associated with risk for new-onset T2D (P = 0.005). Our study demonstrates that a peripheral blood–derived DNA methylation signature is robustly associated with hepatic fat accumulation. The hepatic fat–associated CpGs may represent attractive biomarkers for T2D. Future studies are warranted to explore mechanisms and to examine DNA methylation signatures of NAFLD across racial/ethnic groups.

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.

Amylin, a pancreatic hormone and neuropeptide, acts principally in the hindbrain to decrease food intake and has been recently shown to act as a neurotrophic factor to control the development of AP->NTS and ARC->PVN axonal fiber outgrowth. Amylin is also able to activate ERK signaling specifically in POMC neurons independently of leptin. To investigate the physiological role of amylin signaling in POMC neurons, the core component of the amylin receptor, calcitonin receptor (CTR) was depleted from POMC neurons using an inducible mouse model. The loss of CTR in POMC neurons leads to increased body weight gain, increased adiposity, and glucose intolerance in male knockout mice, characterized by decreased energy expenditure (EE) and decreased expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT). Furthermore, a decreased spontaneous locomotor activity and absent thermogenic reaction to the application of the amylin receptor agonist were observed in male and female mice. Together, these results show a significant physiological impact of amylin/calcitonin signaling in CTR-POMC neurons on energy metabolism and demonstrate the need for sex-specific approaches in obesity research and potentially treatment.

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.

Diabetic retinopathy is a potentially blinding eye disease that threatens the vision of a ninth of diabetic patients. Progression of the disease has long been attributed to an initial dropout of pericytes that enwrap the retinal microvasculature. Revealed through retinal vascular digests, a subsequent increase in basement membrane bridges is observed. Using cell-specific markers, we demonstrate that pericytes rather than endothelial cells colocalize with these bridges. We show that the density of bridges transiently increases with elevation of Ang-2, PDGF-BB, and blood sugar, is rapidly reversed on a time scale of days, and often associated with a pericyte cell body located off-vessel. Cell-specific knockout of KLF4 in pericytes fully replicates this phenotype. In vivo imaging of limbal vessels demonstrates pericyte migration off-vessel, with rapid pericyte filopodial-like process formation between adjacent vessels. Accounting for off-vessel and on-vessel pericytes, we observe no pericyte loss relative to non-diabetic control retina. These findings reveal the possibility that pericyte perturbations in location and process formation may play a role in the development of pathological vascular remodeling in diabetic retinopathy.

A sustained increase in intracellular Ca²⁺ concentration (referred to herein as excitotoxicity), brought on by chronic metabolic stress, may contribute to pancreatic β-cell failure. To determine the additive effects of excitotoxicity and overnutrition on β-cell function and gene expression, we analyzed the impact of a high fat diet (HFD) on Abcc8 knock-out mice. Excitotoxicity caused β-cells to be more susceptible to HFD-induced impairment of glucose homeostasis, and these effects were mitigated by verapamil, a Ca²⁺ channel blocker. Excitotoxicity, overnutrition and the combination of both stresses caused similar but distinct alterations in the β-cell transcriptome, including additive increases in genes associated with mitochondrial energy metabolism, fatty acid β-oxidation and mitochondrial biogenesis, and their key regulator Ppargc1a. Overnutrition worsened excitotoxicity-induced mitochondrial dysfunction, increasing metabolic inflexibility and mitochondrial damage. In addition, excitotoxicity and overnutrition, individually and together, impaired both β-cell function and identity by reducing expression of genes important for insulin secretion, cell polarity, cell junction, cilia, cytoskeleton, vesicular trafficking, and regulation of β-cell epigenetic and transcriptional program. Sex had an impact on all β-cell responses, with male animals exhibiting greater metabolic stress-induced impairments than females. Together, these findings indicate that a sustained increase in intracellular Ca²⁺, by altering mitochondrial function and impairing β-cell identity, augments overnutrition-induced β-cell failure.

Branched chain amino acids (BCAAs) are associated with the progression of obesity-related metabolic disorders, including T2DM and non-alcoholic fatty liver disease. However, whether BCAAs disrupt the homeostasis of hepatic glucose and lipid metabolism remains unknown. In this study, we observed that BCAAs supplementation significantly reduced high-fat (HF) diet-induced hepatic lipid accumulation while increasing the plasma lipid levels and promoting muscular and renal lipid accumulation. Further studies demonstrated that BCAAs supplementation significantly increased hepatic gluconeogenesis and suppressed hepatic lipogenesis in HF diet-induced obese (DIO) mice. These phenotypes resulted from severe attenuation of Akt2 signaling via mTORC1- and mTORC2-dependent pathways. BCAAs/branched-chain α-keto acids (BCKAs) chronically suppressed Akt2 activation through mTORC1 and mTORC2 signaling and promoted Akt2 ubiquitin-proteasome-dependent degradation through the mTORC2 pathway. Moreover, the E3 ligase Mul1 played an essential role in BCAAs/BCKAs-mTORC2-induced Akt2 ubiquitin-dependent degradation. We also demonstrated that BCAAs inhibited hepatic lipogenesis by blocking Akt2/SREBP1/INSIG2a signaling and increased hepatic glycogenesis by regulating Akt2/Foxo1 signaling. Collectively, these data demonstrate that in DIO mice, BCAAs supplementation resulted in serious hepatic metabolic disorder and severe liver insulin resistance: insulin failed to not only suppress gluconeogenesis but also activate lipogenesis. Intervening BCAA metabolism is a potential therapeutic target for severe insulin-resistant disease.

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.

Sodium glucose co-transporter-2 inhibitors (SGLT2i) have favorable cardiovascular outcomes in diabetic patients. However, whether SGLT2i can improve obesity-related cardiac dysfunction is unknown. Sestrin2 is a novel stress-inducible protein that regulates AMPK-mTOR and suppresses oxidative damage. The aim of this study was to determine whether empagliflozin (EMPA) improves obesity-related cardiac dysfunction via regulating Sestrin2-mediated pathways in diet-induced obesity. C57BL/6J mice and Sestrin2 knockout mice were fed a high-fat diet (HFD) for 12 weeks and then treated with or without EMPA (10 mg/kg) for 8 weeks. Treating HFD-fed C57BL/6J mice with EMPA reduced body weight, whole-body fat, and improved metabolic disorders. Furthermore, EMPA improved myocardial hypertrophy/fibrosis and cardiac function, and reduced cardiac fat accumulation and mitochondria injury. Additionally, EMPA significantly augmented Sestrin2 levels, increased AMPK and eNOS phosphorylation, but inhibited Akt and mTOR phosphorylation. These beneficial effects were partially attenuated in HFD-fed Sestrin2 knockout mice. Intriguingly, EMPA treatment enhanced the Nrf2/HO-1-mediated oxidative stress response, suggesting antioxidant and anti-inflammatory activity. Thus, EMPA improved obesity-related cardiac dysfunction via regulating Sestrin2-mediated AMPK-mTOR signaling and maintaining redox homeostasis. These findings provide a novel mechanism for the cardiovascular protection of SGLT2i in obesity.

NADPH facilitates glucose-stimulated insulin secretion (GSIS) in pancreatic islet (PI) β-cells by an as yet unknown mechanism. We found NADPH oxidase, isoform-4 (NOX4), to be the major producer of cytosolic H₂O₂, essential for GSIS, while the increase in ATP/ADP alone was insufficient. The fast GSIS phase was absent in PIs from NOX4-null, β-cell-specific knockout mice (NOX4βKO) (not NOX2KO), and NOX4-silenced or catalase-overexpressing INS-1E cells. Lentiviral NOX4 overexpression or H₂O₂ rescued GSIS in PIs from NOX4βKO mice. NOX4 silencing suppressed Ca²⁺ oscillations and the patch-clamped ATP-sensitive potassium channel (K_ATP) opened more frequently at high glucose. Mitochondrial H₂O₂, decreasing upon GSIS, provided an alternative redox signaling when 2-oxo-isocaproate or fatty acid oxidation formed superoxide by electron-transport flavoprotein:Q-oxidoreductase. Unlike GSIS, this ceased with mitochondrial antioxidant SkQ1. Both NOX4KO and NOX4βKO strains exhibited impaired glucose tolerance and peripheral insulin resistance. Thus the redox signaling previously suggested to cause β-cell-self-checking – hypothetically induces insulin resistance when absent. In conclusion, ATP plus H₂O₂ elevations constitute an essential switch-on signal of insulin exocytosis for glucose and branched-chain oxoacids as secretagogues (partly for fatty acids). Redox signaling could be impaired by cytosolic antioxidants, hence those targeting mitochondria should be preferred for clinical applications to treat (pre)diabetes at any stage.

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.

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.

A greater decrease in 24-h energy expenditure (24EE) during 24h fasting defines a thriftier metabolic phenotype prone to weight gain during overfeeding and resistant to weight loss during caloric restriction. As the thermogenic response to mild cold exposure (COLD) may similarly characterize this human phenotype identified by acute fasting conditions, we analyzed changes in 24EE and sleeping metabolic rate (SLEEP) in a whole-room indirect calorimeter during 24h fasting at thermoneutrality (24°C) and during energy balance both at thermoneutrality (24°C) and mild cold (19°C) in 20 healthy volunteers (80% male, age: 36.6±11.4y, percentage body fat: 34.8±10.5%). Greater decrease in 24EE during fasting (thriftier phenotype) was associated with less increase in 24EE during COLD, i.e. less cold-induced thermogenesis. Greater decreases in plasma fibroblast growth factor 21 (FGF21) after 24h fasting and after COLD were highly correlated and associated with greater decreases in SLEEP in both conditions. We conclude that the metabolic responses to short-term fasting and COLD are associated and mediated by the liver-derived hormone FGF21. Thus, the 24EE response to COLD further identifies the thrifty versus spendthrift phenotype, providing an additional setting to investigate the physiological mechanisms underlying the human metabolic phenotype and characterizing the individual susceptibility to weight change.

Diabetic Keratopathy, a sight-threatening corneal disease, comprises several symptomatic conditions including delayed epithelial wound healing, recurrent erosions, and sensory nerve (SN) neuropathy. We investigated the role of neuropeptides in mediating corneal wound healing, including epithelial wound closure and SN regeneration. Denervation by Resiniferatoxin severely impaired corneal wound healing and markedly up-regulated pro-inflammatory gene expression. Exogenous neuropeptides CGRP, SP, and VIP partially reversed Resiniferatoxin’s effects, with VIP specifically inducing IL-10 expression. Hence, we focused on VIP and observed that wounding induced VIP and VIPR1 expression in normal (NL), but not diabetic (DM) mouse corneas. Targeting VIPR1 in NL corneas attenuated corneal wound healing, dampened wound-induced expression of neurotrophic factors, and exacerbated inflammatory responses while exogenous VIP had the opposite effects in DM corneas. Remarkably, wounding and diabetes also affected the expression of Sonic Hedgehog (SHH) in a VIP-dependent manner. Downregulating SHH expression in NL corneas decreased, while exogenous SHH in DM corneas increased the rates of corneal wound healing. Furthermore, inhibition of SHH signaling dampened VIP-promoted corneal wound healing. We conclude that VIP regulates epithelial wound healing, inflammatory response, and nerve regeneration in the corneas in a SHH-dependent manner, suggesting a therapeutic potential for these molecules in treating diabetic keratopathy.

We conducted a two-sample Mendelian randomisation study to investigate the causal associations of type 2 diabetes mellitus (T2DM) with risk of overall cancer and 22 site-specific cancers. Summary-level data for cancer were extracted from the Breast Cancer Association Consortium and UK Biobank. Genetic predisposition to T2DM was associated with higher odds of pancreatic, kidney, uterine and cervical cancer, lower odds of oesophageal cancer and melanoma, but not associated with 16 other site-specific cancers or overall cancer. The odds ratios (95% confidence interval) were 1.13 (1.04, 1.22), 1.08 (1.00, 1.17), 1.08 (1.01, 1.15), 1.07 (1.01, 1.15), 0.89 (0.81, 0.98), and 0.93 (0.89, 0.97) for pancreatic, kidney, uterine, cervical, and oesophageal cancer and melanoma, respectively. The association between T2DM and pancreatic cancer was also observed in a meta-analysis of this and a previous Mendelian randomisation study (odds ratio 1.08; 1.02, 1.14; p=0.009). There was limited evidence supporting causal associations between fasting glucose and cancer. Genetically predicted fasting insulin levels were positively associated with cancers of the uterus, kidney, pancreas and lung. The present study found causal detrimental effects of T2DM on several cancers. We suggested to reinforce the cancers screening in T2DM patients to enable the early detection of cancer.

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.

Mitochondrial protein FAM3A suppresses hepatic gluconeogenesis and lipogenesis. This study aimed to screen drug(s) that activates FAM3A expression and evaluate its effect(s) on hyperglycemia and steatosis. Drug-repurposing methodology predicted that antidepressive drug doxepin was among the drugs that potentially activated FAM3A expression. Doxepin was further validated to stimulate the translocation of transcription factor HNF4α from the cytoplasm into the nucleus, where it promoted FAM3A transcription to enhance ATP synthesis, suppress gluconeogenesis, and reduce lipid deposition in hepatocytes. HNF4α antagonism or FAM3A deficiency blunted doxepin-induced suppression on gluconeogenesis and lipid deposition in hepatocytes. Doxepin administration attenuated hyperglycemia, steatosis, and obesity in obese diabetic mice with upregulated FAM3A expression in liver and brown adipose tissues (BAT). Notably, doxepin failed to correct dysregulated glucose and lipid metabolism in FAM3A-deficient mice fed on high-fat diet. Doxepin’s effects on ATP production, Akt activation, gluconeogenesis, and lipogenesis repression were also blunted in FAM3A-deficient mouse livers. In conclusion, FAM3A is a therapeutic target for diabetes and steatosis. Antidepressive drug doxepin activates FAM3A signaling pathways in liver and BAT to improve hyperglycemia and steatosis of obese diabetic mice. Doxepin might be preferentially recommended as an antidepressive drug in potential treatment of patients with diabetes complicated with depression.

Milk production may involve a transient development of insulin resistance in non-mammary tissues to support redistribution of maternal macronutrients to match the requirements of the lactating mammary gland. In the present study, adipose and liver metabolic responses were measured in the fasting state and during a 2-step (10 and 20 mU/m²/min) hyperinsulinemic-euglycemic clamp with stable isotopes, in 6-week postpartum women who were lactating (n=12) or formula-feeding (n=6) their infants and who were closely matched for baseline characteristics (e.g., parity, body composition, intrahepatic lipid). When controlling for the low insulin concentrations of both groups, the lactating women exhibited a fasting rate of endogenous glucose production (EGP) that was 2.6-fold greater, and a lipolysis rate that was 2.3-fold greater than the formula-feeding group. During the clamp, the groups exhibited similar suppression rates of EGP and lipolysis. In the lactating women only, higher prolactin concentrations were associated with greater suppression rates of lipolysis, lower intrahepatic lipid and plasma triacylglycerol concentrations. These data suggest that whole-body alterations in glucose transport may be organ specific and facilitate nutrient partitioning during lactation. Recapitulating a shift toward noninsulin-mediated glucose uptake could be an early postpartum strategy to enhance lactation success in women at risk for delayed onset of milk production.

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."

Hepatic steatosis, the excess storage of intrahepatic lipids, is a rampant clinical problem associated with the obesity epidemic. Hepatic steatosis is linked to increased risk for insulin resistance, type 2 diabetes, and cardiovascular and advanced liver disease. Accumulating evidence shows that physical activity, exercise, and aerobic capacity have profound effects on regulating intrahepatic lipids and mediating susceptibility for hepatic steatosis. Moreover, exercise can effectively reduce hepatic steatosis independent of changes in body mass. In this perspective, we highlight 1) the relationship between obesity and metabolic pathways putatively driving hepatic steatosis compared with changes induced by exercise; 2) the impact of physical activity, exercise, and aerobic capacity compared with caloric restriction on regulating intrahepatic lipids and steatosis risk; 3) the effects of exercise training (modalities, volume, intensity) for treatment of hepatic steatosis, and 4) evidence for a sustained protection against steatosis induced by exercise. Overall, evidence clearly indicates that exercise powerfully regulates intrahepatic storage of fat and risk for steatosis.

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.

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.

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.

We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle–dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.

Tumor cells can spread to distant sites through their ability to switch between mesenchymal and amoeboid (bleb-based) migration. Because of this difference, inhibitors of metastasis must account for each migration mode. However, the role of vimentin in amoeboid migration has not been determined. Because amoeboid leader bleb–based migration (LBBM) occurs in confined spaces and vimentin is known to strongly influence cell-mechanical properties, we hypothesized that a flexible vimentin network is required for fast amoeboid migration. To this end, here we determined the precise role of the vimentin intermediate filament system in regulating the migration of amoeboid human cancer cells. Vimentin is a classic marker of epithelial-to-mesenchymal transition and is therefore an ideal target for a metastasis inhibitor. Using a previously developed polydimethylsiloxane slab–based approach to confine cells, RNAi-based vimentin silencing, vimentin overexpression, pharmacological treatments, and measurements of cell stiffness, we found that RNAi-mediated depletion of vimentin increases LBBM by ∼50% compared with control cells and that vimentin overexpression and simvastatin-induced vimentin bundling inhibit fast amoeboid migration and proliferation. Importantly, these effects were independent of changes in actomyosin contractility. Our results indicate that a flexible vimentin intermediate filament network promotes LBBM of amoeboid cancer cells in confined environments and that vimentin bundling perturbs cell-mechanical properties and inhibits the invasive properties of cancer cells.

Motor protein-based active transport is essential for mRNA localization and local translation in animal cells, yet how mRNA granules interact with motor proteins remains poorly understood. Using an unbiased yeast two–hybrid screen for interactions between murine RNA-binding proteins (RBPs) and motor proteins, here we identified protein interaction with APP tail-1 (PAT1) as a potential direct adapter between zipcode-binding protein 1 (ZBP1, a β-actin RBP) and the kinesin-I motor complex. The amino acid sequence of mouse PAT1 is similar to that of the kinesin light chain (KLC), and we found that PAT1 binds to KLC directly. Studying PAT1 in mouse primary hippocampal neuronal cultures from both sexes and using structured illumination microscopic imaging of these neurons, we observed that brain-derived neurotrophic factor (BDNF) enhances co-localization of dendritic ZBP1 and PAT1 within granules that also contain kinesin-I. PAT1 is essential for BDNF-stimulated neuronal growth cone development and dendritic protrusion formation, and we noted that ZBP1 and PAT1 co-locate along with β-actin mRNA in actively transported granules in living neurons. Acute disruption of the PAT1–ZBP1 interaction in neurons with PAT1 siRNA or a dominant-negative ZBP1 construct diminished localization of β-actin mRNA but not of Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) mRNA in dendrites. The aberrant β-actin mRNA localization resulted in abnormal dendritic protrusions and growth cone dynamics. These results suggest a critical role for PAT1 in BDNF-induced β-actin mRNA transport during postnatal development and reveal a new molecular mechanism for mRNA localization in vertebrates.