Aims/hypothesis
Reduced insulin secretion results in hyperglycaemia and diabetes involving a complex aetiology that is yet to be fully elucidated. Genetic susceptibility is a key factor in beta cell ...dysfunction and hyperglycaemia but the responsible genes have not been defined. The Collaborative Cross (CC) is a recombinant inbred mouse panel with diverse genetic backgrounds allowing the identification of complex trait genes that are relevant to human diseases. The aim of this study was to identify and characterise genes associated with hyperglycaemia.
Methods
Using an unbiased genome-wide association study, we examined random blood glucose and insulin sensitivity in 53 genetically unique mouse strains from the CC population. The influences of hyperglycaemia susceptibility quantitative trait loci (QTLs) were investigated by examining glucose tolerance, insulin secretion, pancreatic histology and gene expression in the susceptible mice. Expression of candidate genes and their association with insulin secretion were examined in human islets. Mechanisms underlying reduced insulin secretion were studied in MIN6 cells using RNA interference.
Results
Wide variations in blood glucose levels and the related metabolic traits (insulin sensitivity and body weight) were observed in the CC population. We showed that elevated blood glucose in the CC strains was not due to insulin resistance nor obesity but resulted from reduced insulin secretion. This insulin secretory defect was demonstrated to be independent of abnormalities in islet morphology, beta cell mass and pancreatic insulin content. Gene mapping identified the
E2f8
(
p
= 2.19 × 10
−15
) and
Dlg2
loci (
p
= 3.83 × 10
−8
) on chromosome 7 to be significantly associated with hyperglycaemia susceptibility. Fine mapping the implicated regions using congenic mice demonstrated that these two loci have independent effects on insulin secretion in vivo. Significantly, our results revealed that increased
E2F8
and
DLG2
gene expression are correlated with enhanced insulin secretory function in human islets. Furthermore, loss-of-function studies in MIN6 cells demonstrated that
E2f8
is involved in insulin secretion through an ATP-sensitive K
+
channel-dependent pathway, which leads to a 30% reduction in
Abcc8
expression. Similarly, knockdown of
Dlg2
gene expression resulted in impaired insulin secretion in response to glucose and non-glucose stimuli.
Conclusions/interpretation
Collectively, these findings suggest that E2F transcription factor 8 (E2F8) and discs large homologue 2 (DLG2) regulate insulin secretion. The CC resource enables the identification of
E2f8
and
Dlg2
as novel genes associated with hyperglycaemia due to reduced insulin secretion in pancreatic beta cells. Taken together, our results provide better understanding of the molecular control of insulin secretion and further support the use of the CC resource to identify novel genes relevant to human diseases.
Epigenetic alterations are a hallmark of cancer that govern the silencing of genes. Up to now, 5-azacytidine (5-aza-CR, Vidaza) and 5-aza-2'-deoxycytidine (5-aza-dC, Dacogen) are the only clinically ...approved DNA methyltransferase inhibitors (DNMTi). Current effort tries to exploit DNMTi application beyond acute leukemia or myelodysplastic syndrome, especially to solid tumors. Although both drugs only differ by a minimal structural difference, they trigger distinct molecular mechanisms that are highly relevant for a rational choice of new combination therapies. Therefore, we investigated cell death pathways in vitro in human hepatoma, colon, renal, and lung cancer cells and in vivo in chorioallantoic membrane and xenograft models. Real-time cancer cell monitoring and cytokine profiling revealed a profoundly distinct response pattern to both drugs. 5-aza-dC induced p53-dependent tumor cell senescence and a high number of DNA double-strand breaks. In contrast, 5-aza-CR downregulated p53, induced caspase activation and apoptosis. These individual response patterns of tumor cells could be verified in vivo in chorioallantoic membrane assays and in a hepatoma xenograft model. Although 5-aza-CR and 5-aza-dC are viewed as drugs with similar therapeutic activity, they induce a diverse molecular response in tumor cells. These findings together with other reported differences enable and facilitate a rational design of new combination strategies to further exploit the epigenetic mode of action of these two drugs in different areas of clinical oncology.
Activating JAK2 point mutations are implicated in the pathogenesis of myeloid and lymphoid malignancies, including high-risk B-cell acute lymphoblastic leukemia (B-ALL). In preclinical studies, ...treatment of JAK2 mutant leukemias with type I JAK2 inhibitors (e.g., Food and Drug Administration FDA-approved ruxolitinib) provided limited single-agent responses, possibly due to paradoxical JAK2
hyperphosphorylation induced by these agents. To determine the importance of mutant JAK2 in B-ALL initiation and maintenance, we developed unique genetically engineered mouse models of B-ALL driven by overexpressed Crlf2 and mutant Jak2, recapitulating the genetic aberrations found in human B-ALL. While expression of mutant Jak2 was necessary for leukemia induction, neither its continued expression nor enzymatic activity was required to maintain leukemia survival and rapid proliferation. CRLF2/JAK2 mutant B-ALLs with sustained depletion or pharmacological inhibition of JAK2 exhibited enhanced expression of c-Myc and prominent up-regulation of c-Myc target genes. Combined indirect targeting of c-Myc using the BET bromodomain inhibitor JQ1 and direct targeting of JAK2 with ruxolitinib potently killed JAK2 mutant B-ALLs.
Phagocytosis of apoptotic cells is fundamentally important throughout life, because non-cleared cells become secondarily necrotic and release intracellular contents, thus instigating inflammatory and ...autoimmune responses. Secreted “find-me” and exposed “eat-me” signals displayed by the dying cell in concert with the phagocyte receptors comprise the phagocytic synapse of apoptotic cell clearance. In this scenario, lysophospholipids (lysoPLs) are assumed to act as find-me signals for the attraction of phagocytes. However, both the identity of the lyso-PLs released from apoptotic cells and the nature of the phagocyte receptor are largely unknown. By a detailed analysis of the structural requirements we show here that lysophosphatidylcholine (lysoPC), but none of the lysoPC metabolites or other lysoPLs, represents the essential apoptotic attraction signal able to trigger a phagocyte chemotactic response. Furthermore, using RNA interference and expression studies, we demonstrate that the G-protein-coupled receptor G2A, unlike its relative GPR4, is involved in the chemotaxis of monocytic cells. Thus, our study identifies lysoPC and G2A as the crucial receptor/ligand system for the attraction of phagocytes to apoptotic cells and the prevention of autoimmunity.
Magnetic resonance imaging (MRI) has detected changes in pancreas volume and other characteristics in type 1 and type 2 diabetes. However, differences in MRI technology and approaches across ...locations currently limit the incorporation of pancreas imaging into multisite trials. The purpose of this study was to develop a standardized MRI protocol for pancreas imaging and to define the reproducibility of these measurements. Calibrated phantoms with known MRI properties were imaged at five sites with differing MRI hardware and software to develop a harmonized MRI imaging protocol. Subsequently, five healthy volunteers underwent MRI at four sites using the harmonized protocol to assess pancreas size, shape, apparent diffusion coefficient (ADC), longitudinal relaxation time (T1), magnetization transfer ratio (MTR), and pancreas and hepatic fat fraction. Following harmonization, pancreas size, surface area to volume ratio, diffusion, and longitudinal relaxation time were reproducible, with coefficients of variation less than 10%. In contrast, non-standardized image processing led to greater variation in MRI measurements. By using a standardized MRI image acquisition and processing protocol, quantitative MRI of the pancreas performed at multiple locations can be incorporated into clinical trials comparing pancreas imaging measures and metabolic state in individuals with type 1 or type 2 diabetes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Introduction:
Various functional neuroimaging studies help to better understand the changes in brain activity during meditation. The purpose of this study was to investigate how brain energy ...metabolism changes during focused attention meditation (FAM) state, measured by phosphorous magnetic resonance spectroscopy (
31
P-MRS).
Methods:
31
P-MRS imaging was carried out in 27 participants after 7 weeks of FAM training. Metabolite ratios and the absolute values of metabolites were assessed after meditation training in two MRI measurements, by comparing effects in a FAM state with those in a distinct focused attention awake state during a backwards counting task.
Results:
The results showed decreased phosphocreatine/ATP (PCr/ATP), PCr/ inorganic phosphate (Pi), and intracellular pH values in the entire brain, but especially in basal ganglia, frontal lobes, and occipital lobes, and increased Pi/ATP ratio, cerebral Mg, and Pi absolute values were found in the same areas during FAM compared to the control focused attention awake state.
Conclusions:
Changes in the temporal areas and basal ganglia may be interpreted as a higher energetic state induced by meditation, whereas the frontal and occipital areas showed changes that may be related to a down-regulation in ATP turnover, energy state, and oxidative capacity.
Failure to secrete sufficient quantities of insulin is a pathological feature of type-1 and type-2 diabetes, and also reduces the success of islet cell transplantation. Here we demonstrate that Y1 ...receptor signaling inhibits insulin release in β-cells, and show that this can be pharmacologically exploited to boost insulin secretion. Transplanting islets with Y1 receptor deficiency accelerates the normalization of hyperglycemia in chemically induced diabetic recipient mice, which can also be achieved by short-term pharmacological blockade of Y1 receptors in transplanted mouse and human islets. Furthermore, treatment of non-obese diabetic mice with a Y1 receptor antagonist delays the onset of diabetes. Mechanistically, Y1 receptor signaling inhibits the production of cAMP in islets, which via CREB mediated pathways results in the down-regulation of several key enzymes in glycolysis and ATP production. Thus, manipulating Y1 receptor signaling in β-cells offers a unique therapeutic opportunity for correcting insulin deficiency as it occurs in the pathological state of type-1 diabetes as well as during islet transplantation.Islet transplantation is considered one of the potential treatments for T1DM but limited islet survival and their impaired function pose limitations to this approach. Here Loh et al. show that the Y1 receptor is expressed in β- cells and inhibition of its signalling, both genetic and pharmacological, improves mouse and human islet function.
The engulfment of apoptotic cells is of crucial importance for tissue homeostasis in multicellular organisms. A failure of this process results in secondary necrosis triggering proinflammatory ...cytokine production and autoimmune disease. In the present study, we investigated the role of annexin A1, an intracellular protein that has been implicated in the efficient removal of apoptotic cells. Consistent with its function as bridging protein in the phagocyte synapse, opsonization of apoptotic cells with purified annexin A1 strongly enhanced their phagocytic uptake. A detailed analysis, however, surprisingly revealed that annexin A1 was hardly exposed to the cell surface of primary apoptotic cells, but was strongly externalized only on secondary necrotic cells. Interestingly, while the exposure of annexin A1 failed to promote the uptake of these late secondary necrotic cells, it efficiently prevented induction of cytokine production in macrophages during engulfment of secondary necrotic cells. Our results therefore suggest that annexin A1 exposure during secondary necrosis provides an important failsafe mechanism counteracting inflammatory responses, even when the timely clearance of apoptotic cells has failed.
Background
Meditation is increasingly attracting interest among neuroimaging researchers for its relevance as a cognitive enhancement technique and several cross‐sectional studies have indicated ...cerebral changes. This longitudinal study applied a distinct and standardized meditative technique with a group of volunteers in a short‐term training program to analyze brain metabolic changes.
Methods
The effect of 7 weeks of meditation exercises (focused attention meditation, FAM) was assessed on 27 healthy volunteers. Changes in cerebral energy metabolism were investigated using 31P‐MR spectroscopy. Metabolite ratios were compared before (T1) and after training (T2). Additional questionnaire assessments were included.
Results
The participants performed FAM daily. Depression and anxiety scores revealed a lower level of state anxiety at T2 compared to T1. From T1 to T2, energy metabolism ratios showed the following differences: PCr/ATP increased right occipitally; Pi/ATP decreased bilaterally in the basal ganglia and temporal lobe on the right; PCr/Pi increased in occipital lobe bilaterally, in the basal ganglia and in the temporal lobe on the right side. The pH decreased temporal on the left side and frontal in the right side. The observed changes in the temporal areas and basal ganglia may be interpreted as a higher energetic state, whereas the frontal and occipital areas showed changes that may be related to a down‐regulation in ATP turnover, energy state, and oxidative capacity.
Conclusions
The results of the current study indicate for the first time in a longitudinal study that even short‐term training in FAM may have considerable effects on brain energy state with different local energy management in specific brain regions. Especially higher energetic state in basal ganglia may represent altered function in their central role in complex cerebral distributed networks including frontal and temporal areas. Further studies including different forms of relaxation techniques should be performed for more specific and reliable insights.
Within this MR spectroscopy study, we assessed the effect of 7 weeks of meditation exercises (focused attention meditation, FAM) from 27 healthy volunteers regarding brain metabolic changes. Our results indicate for the first time in a longitudinal study that even short‐term training in FAM has considerable effects on brain energy state in different brain regions.
It is widely accepted that complex interactions between cancer cells and their surrounding microenvironment contribute to disease development, chemo-resistance and disease relapse. In light of this ...observed interdependency, novel therapeutic interventions that target specific cancer stroma cell lineages and their interactions are being sought. Here we studied a mouse model of human T-cell acute lymphoblastic leukaemia (T-ALL) and used intravital microscopy to monitor the progression of disease within the bone marrow at both the tissue-wide and single-cell level over time, from bone marrow seeding to development/selection of chemo-resistance. We observed highly dynamic cellular interactions and promiscuous distribution of leukaemia cells that migrated across the bone marrow, without showing any preferential association with bone marrow sub-compartments. Unexpectedly, this behaviour was maintained throughout disease development, from the earliest bone marrow seeding to response and resistance to chemotherapy. Our results reveal that T-ALL cells do not depend on specific bone marrow microenvironments for propagation of disease, nor for the selection of chemo-resistant clones, suggesting that a stochastic mechanism underlies these processes. Yet, although T-ALL infiltration and progression are independent of the stroma, accumulated disease burden leads to rapid, selective remodelling of the endosteal space, resulting in a complete loss of mature osteoblastic cells while perivascular cells are maintained. This outcome leads to a shift in the balance of endogenous bone marrow stroma, towards a composition associated with less efficient haematopoietic stem cell function. This novel, dynamic analysis of T-ALL interactions with the bone marrow microenvironment in vivo, supported by evidence from human T-ALL samples, highlights that future therapeutic interventions should target the migration and promiscuous interactions of cancer cells with the surrounding microenvironment, rather than specific bone marrow stroma, to combat the invasion by and survival of chemo-resistant T-ALL cells.
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