Receptor-mediated endocytosis, which contributes to a wide range of cellular functions, including receptor signaling, cell adhesion, and migration, requires endocytic vesicle release by the large GTPase dynamin 2. Here, the role of dynamin 2 was investigated in platelet hemostatic function using both pharmacological and genetic approaches. Dnm2^fl/fl Pf4-Cre (Dnm2^Plt–/–) mice specifically lacking dynamin 2 within the platelet lineage developed severe thrombocytopenia and bleeding diathesis and Dnm2^Plt–/– platelets adhered poorly to collagen under arterial shear rates. Signaling via the collagen receptor GPVI was impaired in platelets treated with the dynamin GTPase inhibitor dynasore, as evidenced by poor protein tyrosine phosphorylation, including that of the proximal tyrosine kinase Lyn on its activating tyrosine 396 residue. Platelet stimulation via GPVI resulted in a slight decrease in GPVI, which was maintained by dynasore treatment. Dynasore-treated platelets had attenuated function when stimulated via GPVI, as evidenced by reduced GPIbα downregulation, α-granule release, integrin αIIbβ3 activation, and spreading onto immobilized fibrinogen. By contrast, responses to the G-protein coupled receptor agonist thrombin were minimally affected by dynasore treatment. GPVI expression was severely reduced in Dnm2^Plt–/– platelets, which were dysfunctional in response to stimulation via GPVI, and to a lesser extent to thrombin. Dnm2^Plt–/– platelets lacked fibrinogen in their α-granules, but retained von Willebrand factor. Taken together, the data show that dynamin 2 plays a proximal role in signaling via the collagen receptor GPVI and is required for fibrinogen uptake and normal platelet hemostatic function.

Patients with systemic immunoglobulin light chain amyloidosis (AL) with no evidence of cardiac involvement by consensus criteria have excellent survival, but 20% will die within 5 years of diagnosis and prognostic factors remain poorly characterised. We report the outcomes of 378 prospectively followed Mayo stage I patients (N-terminal pro b-type natriuretic peptide <332 ng/L, high sensitivity cardiac troponin <55 ng/L). The median presenting N-terminal pro b-type natriuretic peptide was 161 ng/L, high sensitivity cardiac troponin 10 ng/L, creatinine 76 μmol/L and mean left ventricular septal wall thickness, 10 mm. Median follow up was 42 (1-117 months), with 71 deaths; median overall survival was not reached (78% survival at 5 years). Although no patients had cardiac involvement by echocardiogram, a proportion (n=25/90, 28%) had cardiac involvement by cardiac magnetic resonance imaging. Age, autonomic nervous system involvement, N-terminal pro b-type natriuretic peptide >152 ng/L, high sensitivity cardiac troponin >10 ng/L and cardiac involvement by magnetic resonance imaging were predictive for survival; on multivariate analysis only N-terminal pro b-type natriuretic peptide >152 ng/L (P<0.008, hazard ratio [HR] 3.180, confidence interval [CI]: 1.349-7.495) and cardiac involvement on magnetic resonance imaging (P=0.026, HR=5.360, CI: 1.219-23.574) were prognostic. At 5 years, 70% of patients with N-terminal pro b-type natriuretic peptide >152 ng/L were alive. In conclusion, N-terminal pro b-type natriuretic peptide is prognostic for survival in patients with no cardiac involvement by consensus criteria and cardiac involvement is detected by magnetic resonance imaging in such cases. This suggests that N-terminal pro b-type natriuretic peptide thresholds for cardiac involvement in AL may need to be redefined.

1q21 amplification is an important prognostic marker in multiple myeloma. In this study we identified that IL6R (the interleukin-6 membrane receptor) and ADAR1 (an RNA editing enzyme) are critical genes located within the minimally amplified 1q21 region. Loss of individual genes caused suppression to the oncogenic phenotypes, the magnitude of which was enhanced when both genes were concomitantly lost. Mechanistically, IL6R and ADAR1 collaborated to induce a hyper-activation of the oncogenic STAT3 pathway. High IL6R confers hypersensitivity to interleukin-6 binding, whereas, ADAR1 forms a constitutive feed-forward loop with STAT3 in a P150-isoform-predominant manner. In this respect, ADAR1-P150 acts as a direct transcriptional target for STAT3 and this STAT3-induced-P150 in turn directly interacts with and stabilizes the former protein, leading to a larger pool of proteins acting as oncogenic transcription factors for pro-survival genes. The importance of both IL6R and ADAR1-P150 in STAT3 signaling was further validated when concomitant knockdown of both genes impeded IL6-induced-STAT3 pathway activation. Clinical evaluation of various datasets of myeloma patients showed that low expression of either one or both genes was closely associated with a compromised STAT3 signature, confirming the involvement of IL6R and ADAR1 in the STAT3 pathway and underscoring their essential role in disease pathogenesis. In summary, our findings highlight the complexity of the STAT3 pathway in myeloma, in association with 1q21 amplification. This study therefore reveals a novel perspective on 1q21 abnormalities in myeloma and a potential therapeutic target for this cohort of high-risk patients.

Tlarge granular lymphocyte leukemia (T-LGLL) is characterized by the expansion of several large granular lymphocyte clones, among which a subset of large granular lymphocytes showing constitutively activated STAT3, a specific CD8⁺/CD4^– phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNAs has not been evaluated in T-LGLL patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. The expression level of 756 mature miRNA was assessed on purified T large granular lymphocytes (T-LGLs) by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-LGLs. Remarkably, CD8 T-LGLs exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of Fas ligand (FasL), that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-LGLs occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-LGLs lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-LGLL.

Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor and together they regulate transcription of actin cytoskeleton genes. MKL1 is associated with hematologic malignancies and immunodeficiency, but its role in B cells is unexplored. Here we examined B cells from monozygotic triplets with an intronic deletion in MKL1, two of whom had been previously treated for Hodgkin lymphoma (HL). To investigate MKL1 and B-cell responses in the pathogenesis of HL, we generated Epstein-Barr virus-transformed lymphoblastoid cell lines from the triplets and two controls. While cells from the patients with treated HL had a phenotype close to that of the healthy controls, cells from the undiagnosed triplet had increased MKL1 mRNA, increased MKL1 protein, and elevated expression of MKL1-dependent genes. This profile was associated with elevated actin content, increased cell spreading, decreased expression of CD11a integrin molecules, and delayed aggregation. Moreover, cells from the undiagnosed triplet proliferated faster, displayed a higher proportion of cells with hyperploidy, and formed large tumors in vivo. This phenotype was reversible by inhibiting MKL1 activity. Interestingly, cells from the triplet treated for HL in 1985 contained two subpopulations: one with high expression of CD11a that behaved like control cells and the other with low expression of CD11a that formed large tumors in vivo similar to cells from the undiagnosed triplet. This implies that pre-malignant cells had re-emerged a long time after treatment. Together, these data suggest that dysregulated MKL1 activity participates in B-cell transformation and the pathogenesis of HL.

Cytogenetic risk stratification at diagnosis has long been one of the most useful tools to assess prognosis in acute lymphoblastic leukemia (ALL). To examine the prognostic impact of cytogenetic abnormalities on outcomes after allogeneic hematopoietic cell transplantation, we studied 1731 adults with Philadelphia-negative ALL in complete remission who underwent myeloablative or reduced intensity/non-myeloablative conditioning transplant from unrelated or matched sibling donors reported to the Center for International Blood and Marrow Transplant Research. A total of 632 patients had abnormal conventional metaphase cytogenetics. The leukemia-free survival and overall survival rates at 5 years after transplantation in patients with abnormal cytogenetics were 40% and 42%, respectively, which were similar to those in patients with a normal karyotype. Of the previously established cytogenetic risk classifications, modified Medical Research Council-Eastern Cooperative Oncology Group score was the only independent prognosticator of leukemia-free survival (P=0.03). In the multivariable analysis, monosomy 7 predicted post-transplant relapse [hazard ratio (HR)=2.11; 95% confidence interval (95% CI): 1.04-4.27] and treatment failure (HR=1.97; 95% CI: 1.20-3.24). Complex karyotype was prognostic for relapse (HR=1.69; 95% CI: 1.06-2.69), whereas t(8;14) predicted treatment failure (HR=2.85; 95% CI: 1.35-6.02) and overall mortality (HR=3.03; 95% CI: 1.44-6.41). This large study suggested a novel transplant-specific cytogenetic scheme with adverse [monosomy 7, complex karyotype, del(7q), t(8;14), t(11;19), del(11q), tetraploidy/near triploidy], intermediate (normal karyotype and all other abnormalities), and favorable (high hyperdiploidy) risks to prognosticate leukemia-free survival (P=0.02). Although some previously established high-risk Philadelphia-negative cytogenetic abnormalities in ALL can be overcome by transplantation, monosomy 7, complex karyotype, and t(8;14) continue to pose significant risks and yield inferior outcomes.

Mixed lineage leukemia (MLL/KMT2A) rearrangements (MLL-r) are one of the most frequent chromosomal aberrations in acute myeloid leukemia. We evaluated the function of Meningioma 1 (MN1), a co-factor of HOXA9 and MEIS1, in human and murine MLL-rearranged leukemia by CRISPR-Cas9 mediated deletion of MN1. MN1 was required for in vivo leukemogenicity of MLL positive murine and human leukemia cells. Loss of MN1 inhibited cell cycle and proliferation, promoted apoptosis and induced differentiation of MLL-rearranged cells. Expression analysis and chromatin immunoprecipitation with sequencing from previously reported data sets demonstrated that MN1 primarily maintains active transcription of HOXA9 and HOXA10, which are critical downstream genes of MLL, and their target genes like BCL2, MCL1 and Survivin. Treatment of MLL-rearranged primary leukemia cells with anti-MN1 siRNA significantly reduced their clonogenic potential in contrast to normal CD34⁺ hematopoietic progenitor cells, suggesting a therapeutic window for MN1 targeting. In summary, our findings demonstrate that MN1 plays an essential role in MLL fusion leukemias and serve as a therapeutic target in MLL-rearranged acute myeloid leukemia.

Autophagy is a genetically regulated process of adaptation to metabolic stress and was recently shown to be involved in the treatment response of chronic myeloid leukemia (CML). However, in vivo data are limited and the molecular mechanism of autophagy regulators in the process of leukemogenesis is not completely understood. Here we show that Beclin-1 knockdown, but not Atg5 deletion in a murine CML model leads to a reduced leukemic burden and results in a significantly prolonged median survival of targeted mice. Further analyses of murine cell lines and primary patient material indicate that active BCR-ABL directly interacts with BECLIN-1 and phosphorylates its tyrosine residues 233 and 352, resulting in autophagy suppression. By using phosphorylation-deficient and phosphorylation-mimic mutants, we identify BCR-ABL induced BECLIN-1 phosphorylation as a crucial mechanism for BECLIN-1 complex formation: interaction analyses exhibit diminished binding of the positive autophagy regulators UVRAG, VPS15, ATG14 and VPS34 and enhanced binding of the negative regulator Rubicon to BCR-ABL-phosphorylated BECLIN-1. Taken together, our findings show interaction of BCR-ABL and BECLIN-1 thereby highlighting the importance of BECLIN-1-mediated autophagy in BCR-ABL⁺ cells.

Mutation and translocation of fibroblast growth factor receptors often lead to aberrant signaling and cancer. This work focuses on the t(8;22)(p11;q11) chromosomal translocation which creates the breakpoint cluster region (BCR) fibroblast growth factor receptor1 (FGFR1) (BCR-FGFR1) fusion protein. This fusion occurs in stem cell leukemia/lymphoma, which can progress to atypical chronic myeloid leukemia, acute myeloid leukemia, or B-cell lymphoma. This work focuses on the biochemical characterization of BCR-FGFR1 and identification of novel therapeutic targets. The tyrosine kinase activity of FGFR1 is required for biological activity as shown using transformation assays, interleukin-3 independent cell proliferation, and liquid chromatography/mass spectroscopy analyses. Furthermore, BCR contributes a coiled-coil oligomerization domain, also essential for oncogenic transformation by BCR-FGFR1. The importance of salt bridge formation within the coiled-coil domain is demonstrated, as disruption of three salt bridges abrogates cellular transforming ability. Lastly, BCR-FGFR1 acts as a client of the chaperonin heat shock protein 90 (Hsp90), suggesting that BCR-FGFR1 relies on Hsp90 complex to evade proteasomal degradation. Transformed cells expressing BCR-FGFR1 are sensitive to the Hsp90 inhibitor Ganetespib, and also respond to combined treatment with Ganetespib plus the FGFR inhibitor BGJ398. Collectively, these data suggest novel therapeutic approaches for future stem cell leukemia/lymphoma treatment: inhibition of BCR oligomerization by disruption of required salt bridges; and inhibition of the chaperonin Hsp90 complex.

Although studies of mixed chimerism following hematopoietic stem cell transplantation in patients with sickle cell disease (SCD) may provide insights into the engraftment needed to correct the disease and into immunological reconstitution, an extensive multilineage analysis is lacking. We analyzed chimerism simultaneously in peripheral erythroid and granulomonocytic precursors/progenitors, highly purified B and T lymphocytes, monocytes, granulocytes and red blood cells (RBC). Thirty-four patients with mixed chimerism and ≥12 months of follow-up were included. A selective advantage of donor RBC and their progenitors/precursors led to full chimerism in mature RBC (despite partial engraftment of other lineages), and resulted in the clinical control of the disease. Six patients with donor chimerism <50% had hemolysis (reticulocytosis) and higher HbS than their donor. Four of them had donor chimerism <30%, including a patient with AA donor (hemoglobin >10 g/dL) and three with AS donors (hemoglobin <10 g/dL). However, only one vaso-occlusive crisis occurred with 68.7% HbS. Except in the patients with the lowest chimerism, the donor engraftment was lower for T cells than for the other lineages. In a context of mixed chimerism after hematopoietic stem cell transplantation for SCD, myeloid (rather than T cell) engraftment was the key efficacy criterion. Results show that myeloid chimerism as low as 30% was sufficient to prevent a vaso-occlusive crisis in transplants from an AA donor but not constantly from an AS donor. However, the correction of hemolysis requires higher donor chimerism levels (i.e. ≥50%) in both AA and AS recipients. In the future, this group of patients may need a different therapeutic approach.

This follow-up study of a randomized, prospective trial included 192 patients with newly diagnosed severe aplastic anemia receiving antithymoglobulin and cyclosporine, with or without granulocyte colony-stimulating factor (G-CSF). We aimed to evaluate the long-term effect of G-CSF on overall survival, event-free survival, probability of secondary myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), clinical paroxysmal nocturnal hemoglobinuria, relapse, avascular osteonecrosis and chronic kidney disease. The median follow-up was 11.7 years (95% CI, 10.9-12.5). The overall survival rate at 15 years was 57±12% in the group given G-CSF and 63±12% in the group not given G-CSF (P=0.92); the corresponding event-free survival rates were 24±10% and 23±10%, respectively (P=0.36). In total, 9 patients developed MDS or AML, 10 only a clonal cytogenetic abnormality, 7 a solid cancer, 18 clinical paroxysmal nocturnal hemoglobinuria, 8 osteonecrosis, and 12 chronic kidney disease, without any difference between patients treated with or without G-CSF. The cumulative incidence of MDS, AML or isolated cytogenetic abnormality at 15 years was 8.5±3% for the G-CSF group and 8.2±3% for the non-G-CSF group (P=0.90). The cumulative incidence of any late event including myelodysplastic syndrome or acute myeloid leukemia, isolated cytogenetic abnormalities, solid cancer, clinical paroxysmal nocturnal hemoglobinuria, aseptic osteonecrosis, chronic kidney disease and relapse was 50±12% for the G-CSF group and 49±12% for the non-G-CSF group (P=0.65). Our results demonstrate that it is unlikely that G-CSF has an impact on the outcome of severe aplastic anemia; nevertheless, very late events are common and eventually affect the prognosis of these patients, irrespectively of their age at the time of immunosuppressive therapy (NCT01163942).

Human bone marrow stromal cells (BMSC) are key elements of the hematopoietic environment and they play a central role in bone and bone marrow physiology. However, how key stromal cell functions are regulated is largely unknown. We analyzed the role of the immediate early response transcription factor EGR1 as key stromal cell regulator and found that EGR1 was highly expressed in prospectively-isolated primary BMSC, down-regulated upon culture, and low in non-colony-forming CD45^neg stromal cells. Furthermore, EGR1 expression was lower in proliferative regenerating adult and fetal primary cells compared to adult steady-state BMSC. Overexpression of EGR1 in stromal cells induced potent hematopoietic stroma support as indicated by an increased production of transplantable CD34⁺CD90⁺ hematopoietic stem cells in expansion co-cultures. The improvement in bone marrow stroma support function was mediated by increased expression of hematopoietic supporting genes, such as VCAM1 and CCL28. Furthermore, EGR1 overexpression markedly decreased stromal cell proliferation whereas EGR1 knockdown caused the opposite effects. These findings thus show that EGR1 is a key stromal transcription factor with a dual role in regulating proliferation and hematopoietic stroma support function that is controlling a genetic program to co-ordinate the specific functions of BMSC in their different biological contexts.

The bone marrow niche is a complex and dynamic structure composed of a multitude of cell types which functionally create an interactive network facilitating hematopoietic stem cell development and maintenance. Its specific role in the pathogenesis, response to therapy, and transformation of myeloproliferative neoplasms has only recently been explored. Niche functionality is likely affected not only by the genomic background of the myeloproliferative neoplasm-associated mutated hematopoietic stem cells, but also by disease-associated ‘chronic inflammation’, and subsequent adaptive and innate immune responses. ‘Cross-talk’ between mutated hematopoietic stem cells and multiple niche components may contribute to propagating disease progression and mediating drug resistance. In this timely article, we will review current knowledge surrounding the deregulated bone marrow niche in myeloproliferative neoplasms and suggest how this may be targeted, either directly or indirectly, potentially influencing therapeutic choices both now and in the future.

Urinary albumin-to-creatinine ratio (ACR) is a marker of diabetic nephropathy and microvascular damage. Metabolic-related traits are observationally associated with ACR, but their causal role is uncertain. Here, we confirmed ACR as a marker of microvascular damage and tested whether metabolic-related traits have causal relationships with ACR. The association between ACR and microvascular function (responses to acetylcholine [ACH] and sodium nitroprusside) was tested in the SUMMIT study. Two-sample Mendelian randomization (MR) was used to infer the causal effects of 11 metabolic risk factors, including glycemic, lipid, and adiposity traits, on ACR. MR was performed in up to 440,000 UK Biobank and 54,451 CKDGen participants. ACR was robustly associated with microvascular function measures in SUMMIT. Using MR, we inferred that higher triglyceride (TG) and LDL cholesterol (LDL-C) levels caused elevated ACR. A 1 SD higher TG and LDL-C level caused a 0.062 (95% CI 0.040, 0.083) and a 0.026 (95% CI 0.008, 0.044) SD higher ACR, respectively. There was evidence that higher body fat and visceral body fat distribution caused elevated ACR, while a metabolically "favorable adiposity" phenotype lowered ACR. ACR is a valid marker for microvascular function. MR suggested that seven traits have causal effects on ACR, highlighting the role of adiposity-related traits in causing lower microvascular function.

Diabetic keratopathy occurs in ~70% of all people with diabetes. This study was designed to examine the effects of vitamin D receptor knockout (VDR^–/–) and vitamin D deficiency (VDD) on corneal epithelial wound healing and nerve density in diabetic mice. Diabetes was induced using the low-dose streptozotocin method. Corneal epithelial wounds were created using an Algerbrush, and wound healing was monitored over time. Corneal nerve density was measured in unwounded mice. VDR^–/– and VDD diabetic mice (diabetic for 8 and 20 weeks, respectively) had slower healing ratios than wild-type diabetic mice. VDR^–/– and VDD diabetic mice also showed significantly decreased nerve density. Reduced wound healing ratios and nerve densities were not fully rescued by a supplemental diet rich in calcium, lactose, and phosphate. We conclude that VDR^–/– and VDD significantly reduce both corneal epithelial wound healing and nerve density in diabetic mice. Because the supplemental diet did not rescue wound healing or nerve density, these effects are likely not specifically related to hypocalcemia. This work supports the hypothesis that low vitamin D levels can exacerbate preexisting ophthalmic conditions, such as diabetes.

Type 2 diabetes (T2D) is caused by loss of pancreatic β-cell mass and failure of the remaining β-cells to deliver sufficient insulin to meet demand. β-Cell glucolipotoxicity (GLT), which refers to combined, deleterious effects of elevated glucose and fatty acid levels on β-cell function and survival, contributes to T2D-associated β-cell failure. Drugs and mechanisms that protect β-cells from GLT stress could potentially improve metabolic control in patients with T2D. In a phenotypic screen seeking low-molecular-weight compounds that protected β-cells from GLT, we identified compound A that selectively blocked GLT-induced apoptosis in rat insulinoma cells. Compound A and its optimized analogs also improved viability and function in primary rat and human islets under GLT. We discovered that compound A analogs decreased GLT-induced cytosolic calcium influx in islet cells, and all measured β-cell–protective effects correlated with this activity. Further studies revealed that the active compound from this series largely reversed GLT-induced global transcriptional changes. Our results suggest that taming cytosolic calcium overload in pancreatic islets can improve β-cell survival and function under GLT stress and thus could be an effective strategy for T2D treatment.

The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study demonstrated that intensive glucose control reduced the risk of developing diabetic peripheral neuropathy (DPN) and cardiovascular autonomic neuropathy (CAN). We evaluated multiple risk factors and phenotypes associated with DPN and CAN in this large, well-characterized cohort of participants with type 1 diabetes, followed for >23 years. DPN was defined by symptoms, signs, and nerve conduction study abnormalities in ≥2 nerves; CAN was assessed using standardized cardiovascular reflex tests. Generalized estimating equation models assessed the association of DPN and CAN with individual risk factors measured repeatedly. During DCCT/EDIC, 33% of participants developed DPN and 44% CAN. Higher mean HbA_1c was the most significant risk factor for DPN, followed by older age, longer duration, greater height, macroalbuminuria, higher mean pulse rate, β-blocker use, and sustained albuminuria. The most significant risk factor for CAN was older age, followed by higher mean HbA_1c, sustained albuminuria, longer duration of type 1 diabetes, higher mean pulse rate, higher mean systolic blood pressure, β-blocker use, estimated glomerular filtration rate <60 mL/min/1.73 m², higher most recent pulse rate, and cigarette smoking. These findings identify risk factors and phenotypes of participants with diabetic neuropathy that can be used in the design of new interventional trials and for personalized approaches to neuropathy prevention.

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin (PI) in the endoplasmic reticulum (ER) drive the molecular pathogenesis of mutant INS gene–induced diabetes of youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, PI-WT dimerizes in the ER. Here, we suggest that the normal PI-PI contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that PI B-chain tyrosine-16 (Tyr-B16), which is a key residue in normal PI dimerization, helps confer dominant-negative behavior of MIDY mutant PI-C(A7)Y. Substitutions of Tyr-B16 with either Ala, Asp, or Pro in PI-C(A7)Y decrease the abnormal interactions between the MIDY mutant and PI-WT, rescuing PI-WT export, limiting ER stress, and increasing insulin production in β-cells and human islets. This study reveals the first evidence indicating that noncovalent PI-PI contact initiates dominant-negative behavior of misfolded PI, pointing to a novel therapeutic target to enhance PI-WT export and increase insulin production.

Vitamin D deficiency has been associated with increased incidence of diabetes, both in humans and in animal models. In addition, an association between vitamin D receptor (VDR) gene polymorphisms and diabetes has also been described. However, the involvement of VDR in the development of diabetes, specifically in pancreatic β-cells, has not been elucidated yet. Here, we aimed to study the role of VDR in β-cells in the pathophysiology of diabetes. Our results indicate that Vdr expression was modulated by glucose in healthy islets and decreased in islets from both type 1 diabetes and type 2 diabetes mouse models. In addition, transgenic mice overexpressing VDR in β-cells were protected against streptozotocin-induced diabetes and presented a preserved β-cell mass and a reduction in islet inflammation. Altogether, these results suggest that sustained VDR levels in β-cells may preserve β-cell mass and β-cell function and protect against diabetes.

Genes of the Notch signaling pathway are expressed in different cell types and organs at different time points during embryonic development and adulthood. The Notch ligand Delta-like 1 (DLL1) controls the decision between endocrine and exocrine fates of multipotent progenitors in the developing pancreas, and loss of Dll1 leads to premature endocrine differentiation. However, the role of Delta-Notch signaling in adult tissue homeostasis is not well understood. Here, we describe the spatial expression pattern of Notch pathway components in adult murine pancreatic islets and show that DLL1 and DLL4 are specifically expressed in β-cells, whereas JAGGED1 is expressed in α-cells. We show that mice lacking both DLL1 and DLL4 in adult β-cells display improved glucose tolerance, increased glucose-stimulated insulin secretion, and hyperglucagonemia. In contrast, overexpression of the intracellular domain of DLL1 in adult murine pancreatic β-cells results in impaired glucose tolerance and reduced insulin secretion, both in vitro and in vivo. These results suggest that Notch ligands play specific roles in the adult pancreas and highlight a novel function of the Delta/Notch pathway in β-cell insulin secretion.

An aging global population combined with sedentary lifestyles and unhealthy diets has contributed to an increasing incidence of obesity and type 2 diabetes. These metabolic disorders are associated with perturbations to nitric oxide (NO) signaling and impaired glucose metabolism. Dietary inorganic nitrate, found in high concentration in green leafy vegetables, can be converted to NO in vivo and demonstrates antidiabetic and antiobesity properties in rodents. Alongside tissues including skeletal muscle and liver, white adipose tissue is also an important physiological site of glucose disposal. However, the distinct molecular mechanisms governing the effect of nitrate on adipose tissue glucose metabolism and the contribution of this tissue to the glucose-tolerant phenotype remain to be determined. Using a metabolomic and stable-isotope labeling approach, combined with transcriptional analysis, we found that nitrate increases glucose uptake and oxidative catabolism in primary adipocytes and white adipose tissue of nitrate-treated rats. Mechanistically, we determined that nitrate induces these phenotypic changes in primary adipocytes through the xanthine oxidoreductase–catalyzed reduction of nitrate to NO and independently of peroxisome proliferator–activated receptor-α. The nitrate-mediated enhancement of glucose uptake and catabolism in white adipose tissue may be a key contributor to the antidiabetic effects of this anion.

Glucagon is classically described as a counterregulatory hormone that plays an essential role in the protection against hypoglycemia. In addition to its role in the regulation of glucose metabolism, glucagon has been described to promote ketosis in the fasted state. Sodium–glucose cotransporter 2 inhibitors (SGLT2i) are a new class of glucose-lowering drugs that act primarily in the kidney, but some reports have described direct effects of SGLT2i on α-cells to stimulate glucagon secretion. Interestingly, SGLT2 inhibition also results in increased endogenous glucose production and ketone production, features common to glucagon action. Here, we directly test the ketogenic role of glucagon in mice, demonstrating that neither fasting- nor SGLT2i-induced ketosis is altered by interruption of glucagon signaling. Moreover, any effect of glucagon to stimulate ketogenesis is severely limited by its insulinotropic actions. Collectively, our data suggest that fasting-associated ketosis and the ketogenic effects of SGLT2 inhibitors occur almost entirely independent of glucagon.

Subjects with low serum HDL cholesterol levels are reported to be susceptible to diabetes, with insulin resistance believed to be the underlying pathological mechanism. Apolipoprotein M (apoM) is a carrier of sphingosine-1-phosphate (S1P), a multifunctional lipid mediator, on HDL, and the pleiotropic effects of HDL are believed to be mediated by S1P. In the current study, we attempted to investigate the potential association between apoM/S1P and insulin resistance. We observed that the serum levels of apoM were lower in patients with type 2 diabetes and that they were negatively correlated with BMI and the insulin resistance index. While deletion of apoM in mice was associated with worsening of insulin resistance, overexpression of apoM was associated with improvement of insulin resistance. Presumably, apoM/S1P exerts its protective effect against insulin resistance by activating insulin signaling pathways, such as the AKT and AMPK pathways, and also by improving the mitochondrial functions through upregulation of SIRT1 protein levels. These actions of apoM/S1P appear to be mediated via activation of S1P1 and/or S1P3. These results suggest that apoM/S1P exerts protective roles against the development of insulin resistance.

Accumulation of lipid in skeletal muscle is thought to be related to the development of insulin resistance and type 2 diabetes. Initial work in this area focused on accumulation of intramuscular triglyceride; however, bioactive lipids such as diacylglycerols and sphingolipids are now thought to play an important role. Specific species of these lipids appear to be more negative toward insulin sensitivity than others. Adding another layer of complexity, localization of lipids within the cell appears to influence the relationship between these lipids and insulin sensitivity. This article summarizes how accumulation of total lipids, specific lipid species, and localization of lipids influence insulin sensitivity in humans. We then focus on how these aspects of muscle lipids are impacted by acute and chronic aerobic and resistance exercise training. By understanding how exercise alters specific species and localization of lipids, it may be possible to uncover specific lipids that most heavily impact insulin sensitivity.

Insulin resistance is an underappreciated facet of type 1 diabetes that occurs with remarkable consistency and considerable magnitude. Although therapeutic innovations are continuing to normalize dysglycemia, a sizable body of data suggests a second metabolic abnormality—iatrogenic hyperinsulinemia—principally drives insulin resistance and its consequences in this population and has not been addressed. We review this evidence to show that injecting insulin into the peripheral circulation bypasses first-pass hepatic insulin clearance, which leads to the unintended metabolic consequence of whole-body insulin resistance. We propose restructuring insulin therapy to restore the physiological insulin balance between the hepatic portal and peripheral circulations and thereby avoid the complications of life-long insulin resistance. As technology rapidly advances and our ability to ensure euglycemia improves, iatrogenic insulin resistance will become the final barrier to overcome to restore normal physiology, health, and life in type 1 diabetes.

Advances in small RNA sequencing have revealed the enormous diversity of small noncoding RNA (sRNA) classes in mammalian cells. At this point, most investigators in diabetes are aware of the success of microRNA (miRNA) research and appreciate the importance of posttranscriptional gene regulation in glycemic control. Nevertheless, miRNAs are just one of multiple classes of sRNAs and likely represent only a minor fraction of sRNA sequences in a given cell. Despite the widespread appreciation of sRNAs, very little research into non-miRNA sRNA function has been completed, likely due to some major barriers that present unique challenges for study. To emphasize the importance of sRNA research in cardiometabolic diseases, we highlight the success of miRNAs and competitive endogenous RNAs in cholesterol and glucose metabolism. Moreover, we argue that sequencing studies have demonstrated that miRNAs are just the tip of the iceberg for sRNAs. We are likely standing at the precipice of immense discovery for novel sRNA-mediated gene regulation in cardiometabolic diseases. To realize this potential, we must first address critical barriers with an open mind and refrain from viewing non-miRNA sRNA function through the lens of miRNAs, as they likely have their own set of distinct regulatory factors and functional mechanisms.

Nayeli G. Reyes-Nava, Hung-Chun Yu, Curtis R. Coughlin II, Tamim H. Shaikh, and Anita M. Quintana

We used whole-exome sequencing (WES) to determine the genetic etiology of a patient with a multi-system disorder characterized by a seizure phenotype. WES identified a heterozygous de novo missense mutation in the GABRA1 gene (c.875C>T). GABRA1 encodes the alpha subunit of the gamma-aminobutyric acid receptor A (GABA_AR). The GABA_AR is a ligand gated ion channel that mediates the fast inhibitory signals of the nervous system, and mutations in the subunits that compose the GABA_AR have been previously associated with human disease. To understand the mechanisms by which GABRA1 regulates brain development, we developed a zebrafish model of gabra1 deficiency. gabra1 expression is restricted to the nervous system and behavioral analysis of morpholino injected larvae suggests that the knockdown of gabra1 results in hypoactivity and defects in the expression of other subunits of the GABA_AR. Expression of the human GABRA1 protein in morphants partially restored the hypomotility phenotype. In contrast, the expression of the c.875C>T variant did not restore these behavioral deficits. Collectively, these results represent a functional approach to understand the mechanisms by which loss-of-function alleles cause disease.

Yuta Sakae, Akira Oikawa, Yuki Sugiura, Masatoshi Mita, Shuhei Nakamura, Toshiya Nishimura, Makoto Suematsu, and Minoru Tanaka

The teleost fish, medaka (Oryzias latipes), employs the XX/XY genetic sex determination system. We show here that the phenotypic sex of medaka is affected by changes in lipid metabolism. Medaka larvae subjected to 5 days of starvation underwent female-to-male sex reversal. Metabolomic and RT-qPCR analyses indicated that pantothenate metabolism was suppressed by starvation. Consistently, inhibiting the pantothenate metabolic pathway caused sex reversal. The final metabolite in this pathway is coenzyme A, an essential factor for lipogenesis. Inhibiting fatty acid synthesis, the first step of lipogenesis, also caused sex reversal. The expression of dmrt1, a critical gene for male development, was suppressed by starvation, and a dmrt1 (13) mutant did not show sex reversal under starvation. Collectively, these results indicate that fatty acid synthesis is involved in female-to-male sex reversal through ectopic expression of male gene dmrt1 under starvation.

BackgroundAutistic people are at increased risk of developing mental health problems. To reduce the negative impact of living with autism in a non-autistic world, efforts to improve take-up and access to care, and support in early years, which will typically start with a GP appointment, must be grounded in the accounts of autistic young adults.AimTo explore how autistic young adults understand and manage mental health problems; and to consider help seeking as a focus.Design and settingA cross-sectional, qualitative study. Autistic participants were purposively selected to represent a range of mental health conditions including anxiety and depression. A subsample were recruited from a population cohort screened for autism in childhood. The study concerns access to primary care.MethodNineteen autistic young adults without learning disabilities, aged 23 or 24 years, were recruited. In-depth, semi-structured interviews explored how they understood and managed mental health problems. Data were analysed thematically.ResultsYoung adults preferred self-management strategies. Multiple factors contributed to a focus on self-management, including: beliefs about the aetiology of mental health difficulties and increased vulnerability with the context of a diagnosis of autism, knowledge of self-management, and a view that formal support was unavailable or inadequate. Families had limited awareness of professional support.ConclusionYoung autistic adults without learning disabilities, and their families, may hold erroneous beliefs about autism and mental health. This may affect help seeking and contribute to an exacerbation of symptoms. GPs need to be alert to the fact that autistic young adults in their care may be experiencing mental health difficulties but may not recognise them as such.

BackgroundUrinary tract infections (UTIs) are one of the most common bacterial infections managed in general practice. Many women with symptoms of uncomplicated UTI may not benefit meaningfully from antibiotic treatment, but the evidence base is complex and there is no suitable shared decision-making resource to guide antibiotic treatment and symptomatic care for use in general practice consultations.AimTo develop an evidence-based, shared decision-making intervention leaflet to optimise management of uncomplicated UTI for women aged <65 years in the primary care setting.Design and settingQualitative telephone interviews with GPs and patient focus group interviews.MethodIn-depth interviews were conducted to explore how consultation discussions around diagnosis, antibiotic use, self-care, safety netting, and prevention of UTI could be improved. Interview schedules were based on the Theoretical Domains Framework.ResultsBarriers to an effective joint consultation and appropriate prescribing included: lack of GP time, misunderstanding of depth of knowledge and miscommunication between the patient and the GP, nature of the consults (such as telephone consultations), and a history of previous antibiotic therapy.ConclusionConsultation time pressures combined with late symptom presentation are a challenge for even the most experienced of GPs: however, it is clear that enhanced patient–clinician shared decision making is urgently required when it comes to UTIs. This communication should incorporate the provision of self-care, safety netting, and preventive advice to help guide patients when to consult. A shared decision-making information leaflet was iteratively co-produced with patients, clinicians, and researchers at Public Health England using study data.

BackgroundInfection is common in older adults. Serious infection has a high mortality rate and is associated with unplanned hospital admissions. Little is known about the factors that prompt older patients to seek medical advice when they may have an infection.AimTo explore the symptoms of infection from the perspective of older adults, and when and why older patients seek healthcare advice for a possible infection.Design and settingA qualitative interview study among adults aged ≥70 years with a clinical diagnosis of infection recruited from ambulatory care units in Oxford, UK.MethodInterviews were semi-structured and based on a flexible topic guide. Participants were given the option to be interviewed with their carer. Thematic analysis was facilitated using NVivo (version 11).ResultsA total of 28 participants (22 patients and six carers) took part. Patients (aged 70–92 years) had experienced a range of different infections. Several early non-specific symptoms were described (fever, feeling unwell, lethargy, vomiting, pain, and confusion/delirium). Internally minimising symptoms was common and participants with historical experience of infection tended to be better able to interpret their symptoms. Factors influencing seeking healthcare advice included prompts from family, specific or intolerable symptoms, symptom duration, and being unable to manage with self-care. For some, not wanting to be a burden affected their desire to seek help.ConclusionTailored advice to older adults highlighting early symptoms of infection may be beneficial. Knowing whether patients have had previous experience of infection may help healthcare professionals in assessing older patients with possible infection.

BackgroundIn the context of a variable condition such as asthma, patient recognition of deteriorating control and knowing what prompt action to take is crucial. Yet, implementation of recommended self-management strategies remains poor.AimTo explore how patients with asthma and parents/carers of children with asthma develop and establish recommended self-management strategies for living with asthma, and how clinicians can best support the process.Design and settingA qualitative study in UK primary care.MethodPatients with asthma and parents/carers of children with asthma from 10 general practices were purposively sampled (using age, sex, and duration of asthma) to participate in focus groups or interviews between May 2016 and August 2016. Participants’ experiences of health care, management of asthma, and views on supported self-management were explored. Interviews and focus group sessions were audio-recorded and transcribed verbatim. Iterative thematic analysis was conducted, guided by the research questions and drawing on habit theory in discussion with a multidisciplinary research team.ResultsA total of 49 participants (45 patients; 4 parents/carers) took part in 32 interviews and five focus groups. Of these, 11 reported using an action plan. Patients learnt how to self-manage over time, building knowledge from personal experience and other sources, such as the internet. Some regular actions, for example, taking medication, became habitual. Dealing with new or unexpected scenarios required reflective abilities, which may be supported by a tailored action plan.ConclusionPatients reported learning intuitively how to self-manage. Some regular actions became habitual; dealing with the unexpected required more reflective cognitive skills. In order to support implementation of optimal asthma self- management, clinicians should consider both these aspects of self-management and support, and educate patients proactively.