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Aims/hypothesis Pancreatic beta cells are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce reactive oxygen species to protect against apoptosis. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesised that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development. Methods Pancreases were collected from chloroquine-injected and non-injected non-obese diabetes-resistant (NOR) and non-obese diabetic (NOD) mice. Age- and BMI-matched pancreas tissue sections from human organ donors (N = 34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), microtubule-associated protein 1 light chain 3 A/B (LC3A/B; autophagosome marker), lysosomal-associated membrane protein 1 (LAMP1; lysosome marker) and p62 (autophagy adaptor). Images collected on a scanning laser confocal microscope were analysed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes were assessed in electron micrographs of human pancreatic tissue sections (n = 12), and energy dispersive x-ray analysis was performed to assess distribution of elements (n = 5). Results We observed increased autophagosome numbers in islets of diabetic NOD mice (p = 0.008) and increased p62 in islets of both non-diabetic and diabetic NOD mice (p p p p = 0.003) and non-diabetic NOD mice (p p p p p = 0.002). Conclusions/interpretation We provide evidence of islet macroautophagy/crinophagy impairment in human type 1 diabetes. We also document accumulation of telolysosomes with peripheral nitrogen in beta cells of autoantibody-positive donors, demonstrating altered lysosome content that may be associated with lysosome dysfunction before clinical hyperglycaemia. Similar macroautophagy impairments are present in the NOD mouse model of type 1 diabetes. Graphical abstract

In type 1 diabetes, an autoimmune event increases oxidative stress in islet β cells, giving rise to cellular dysfunction and apoptosis. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids that can form lipid metabolites involved in several biological functions, including oxidative stress. 12-Lipoxygenase and 12/15-lipoxygenase are related but distinct enzymes that are expressed in pancreatic islets, but their relative contributions to oxidative stress in these regions are still being elucidated. In this study, we used mice with global genetic deletion of the genes encoding 12-lipoxygenase (arachidonate 12-lipoxygenase, 12S type [Alox12]) or 12/15-lipoxygenase (Alox15) to compare the influence of each gene deletion on β cell function and survival in response to the β cell toxin streptozotocin. Alox12(−/−) mice exhibited greater impairment in glucose tolerance following streptozotocin exposure than WT mice, whereas Alox15(−/−) mice were protected against dysglycemia. These changes were accompanied by evidence of islet oxidative stress in Alox12(−/−) mice and reduced oxidative stress in Alox15(−/−) mice, consistent with alterations in the expression of the antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12(−/−) mice displayed a compensatory increase in Alox15 gene expression, and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. Collectively, these results indicate that Alox12 loss activates a compensatory increase in Alox15 that sensitizes mouse β cells to oxidative stress.

A large body of evidence reveals a web of complex, multifaceted genetic, and environmental factors that influences incidence of T1D. Among these, viral infection has been discussed as a factor implicated in onset. Production of type 1 interferons (IFN) is the archetypal immune response to viral infection. Indeed, at-risk children exhibit IFN-associated transcriptional profiles and IFN-stimulated genes are elevated in new onset T1D patients. IFN-α induces ER stress, substantial overexpression of HLA class 1, and insulitis in the islet. β-cells are known to be sensitive to reactive oxygen species (ROS) accumulation which may contribute to autoimmunogenicity in the IFN response. In order to detect the in vivo effects of IFN-α on the physiology of human islets, we conducted intravital microscopy studies of human islets transplanted in the mouse kidney - following transduction with a ratiometric fluorescent redox biosensor. These studies revealed that intravenous IFN-α elicited a mild overall accumulation of ROS in human islets, but rapid and pronounced accumulation of ROS in a subset of islet β-cells - a population of cells we termed, “ROSponders." Interestingly, the percentage of β-cells categorized as ROSponders was variable between donors (ranging from ~1% to 6% of all β-cells). Recent observations have drawn attention to the importance of β-cell expression and functional heterogeneity within the islet. Furthermore, recent work suggests that blockade of IFN-α signaling prior to clinical T1D manifestation may intercept autoimmunity and abort onset. Altogether, this work identifies novel heterogeneity of response to an inflammatory cytokine implicated in T1D onset and may inform future approaches to the treatment of T1D. Disclosure M. Arvin: None. C. Muralidharan: None. A. M. Conteh: None. M. M. Martinez irizarry: None. K. Orr: None. K. W. Dunn: None. A. K. Linnemann: None. Funding Human Islet Research Network (RRID:SCR_014393; UC4 DK104162); National Institutes of Health (3T32DK064466-18S1)

Aims/hypothesisPancreatic beta cells are highly metabolic secretory cells that are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species (ROS) during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce ROS to protect against apoptosis both in vitro and in vivo. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesized that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development.MethodsMouse pancreata were collected from chloroquine injected and non-injected NOR, nondiabetic NOD, and diabetic NOD mice. Age and BMI-matched pancreas tissue sections from human organ donors (n=34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). To assess autophagic flux, we injected the mice with chloroquine to inhibit the final stages of autophagy. We analyzed tissues for markers of autophagy via immunofluorescence analysis. Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), LC3A/B (autophagosome marker), Lamp1 (lysosome marker), and p62 (autophagy adaptor protein and marker for autophagic flux). Images were collected on a scanning laser confocal microscope then analyzed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes (formerly called lipofuscin bodies, residual bodies or tertiary lysosomes) were analyzed in electron micrographs of pancreatic tissue sections from human organ donors (nPOD; n=12) deposited in www.nanotomy.org/OA/nPOD. Energy Dispersive X-ray (EDX) analysis was also performed on these tissues to analyze distribution of elements such as nitrogen, phosphorus, and osmium in secondary lysosomes and telolysosomes of nondiabetic and autoantibody positive donor tissues (n=5).ResultsWe observed increased autophagosome numbers in islets of diabetic NOD mice (p=0.0077) and increased p62 in islets of both nondiabetic and diabetic NOD mice (pConclusions/interpretationCollectively, we provide evidence of impairment in the final degradation stages of islet macroautophagy and crinophagy in human type 1 diabetes. We also document an accumulation of telolysosomes with nitrogen accumulation at their periphery in the beta cells of autoantibody positive donors. This demonstrates clear differences in the lysosome contents of autoantibody positive donors that may be associated with lysosome dysfunction prior to clinical hyperglycemia. We observe similar impairments in macroautophagy in the diabetic NOD mouse, a model of type 1 diabetes, suggesting that this mouse model can be appropriately used to study the pathogenesis of autophagy/crinophagy loss and how it relates to disease initiation and progression. Considering these data in the context of what is known regarding the cell-protective effects of islet autophagy, we suggest targeting beta cell autophagy pathways as an approach to reduce apoptosis in individuals at risk for type 1 diabetes development.Research in contextWhat is already known about this subject?Autophagy confers a cytoprotective role in the beta cell.Autophagy is reduced in type 2 diabetes.Autophagy in the context of type 1 diabetes is unexplored.What is the key question?Is autophagy reduced during the pathogenesis of human type 1 diabetes?What are the new findings?We provide evidence of reduced autophagy and crinophagy in human type 1 diabetes.These data are supported by observations of reduced autophagy in a mouse model of autoimmune diabetes.How might this impact on clinical practice in the foreseeable future?This study provides evidence that autophagy is impaired in human type 1 diabetes. Prior studies have shown that loss of autophagy in the islet is associated with increased beta cell apoptosis, therefore we propose that therapeutic targeting of autophagy pathways may reduce beta cell death in type 1 diabetes development.

Autophagy is a dynamic recycling mechanism that maintains beta cell homeostasis, whereas the related process of crinophagy also plays a specific role in insulin granule regulation. Impairment of autophagy and crinophagy have been implicated in beta cell death and dysfunction in the context of type 2 diabetes; however, there is currently no literature describing beta cell autophagy in type 1 diabetes pathogenesis. Therefore, we set out to determine if beta cell autophagy and crinophagy are impaired in the context of type 1 diabetes. We utilized immunofluorescent staining of LC3 (autophagosome marker), Lamp1 (lysosome marker), and proinsulin to analyze pancreatic islet autophagy and crinophagy in the NOD (non-obese diabetic) mouse model of type 1 diabetes as well as in human pancreas tissue obtained from the network for Pancreatic Organ donors with Diabetes (nPOD). We observed evidence of reduced macroautophagy in the residual proinsulin-positive islet cells of human donors with type 1 diabetescompared to controls, as measured by significantly reduced autophagosome:lysosome colocalization (p=0.034). Similarly, there was a trend towards reduced autophagosome:lysosome colocalization in islets of diabetic NOD mice compared to nondiabetic NOR mice (p=0.08). We also observed significantly reduced colocalization of proinsulin with lysosomes in the proinsulin positive cells from human donors with type 1 diabetes in comparison to both controls without diabetes (p=0.0014) and autoantibody positive donors (p= 0.0278), as well as in diabetic NOD vs. nondiabetic NOR mice (p=0.0121), suggesting that crinophagy is also reduced as a function of type 1 diabetes. Collectively, we provide evidence that macroautophagy and crinophagy are reduced in the context of type 1 diabetes. Considering these data in the context of what is known regarding the cell-protective effects of islet autophagy, we suggest targeting beta cell autophagy pathways as an approach to reduce apoptosis in individuals at risk for type 1 diabetes development. Disclosure C. Muralidharan: None. M. Marasco: Employee; Spouse/Partner; Eli Lilly and Company. J. Crowder: Employee; Spouse/Partner; Eli Lilly and Company. A.M. Conteh: None. A.K. Linnemann: None.

Production of reactive oxygen species (ROS) is a key instigator of β-cell dysfunction in diabetes. The pleiotropic cytokine interleukin 6 (IL-6) has previously been linked to β-cell autophagy but has not been studied in the context of β-cell antioxidant response. We used a combination of animal models of diabetes and analysis of cultured human islets and rodent β-cells to study how IL-6 influences antioxidant response. We show that IL-6 couples autophagy to antioxidant response and thereby reduces ROS in β-cells and human islets. β-Cell-specific loss of IL-6 signaling in vivo renders mice more susceptible to oxidative damage and cell death through the selective β-cell toxins streptozotocin and alloxan. IL-6-driven ROS reduction is associated with an increase in the master antioxidant factor NRF2, which rapidly translocates to the mitochondria to decrease mitochondrial activity and stimulate mitophagy. IL-6 also initiates a robust transient decrease in cellular cAMP levels, likely contributing to the stimulation of mitophagy to mitigate ROS. Our findings suggest that coupling autophagy to antioxidant response in β-cells leads to stress adaptation that can reduce cellular apoptosis. These findings have implications for β-cell survival under diabetogenic conditions and present novel targets for therapeutic intervention.

Islet β-cell dysfunction and aggressive macrophage activity are early features in the pathogenesis of type 1 diabetes (T1D). 12/15-Lipoxygenase (12/15-LOX) is induced in β-cells and macrophages during T1D and produces proinflammatory lipids and lipid peroxides that exacerbate β-cell dysfunction and macrophage activity. Inhibition of 12/15-LOX provides a potential therapeutic approach to prevent glycemic deterioration in T1D. Two inhibitors recently identified by our groups through screening efforts, ML127 and ML351, have been shown to selectively target 12/15-LOX with high potency. Only ML351 exhibited no apparent toxicity across a range of concentrations in mouse islets, and molecular modeling has suggested reduced promiscuity of ML351 compared with ML127. In mouse islets, incubation with ML351 improved glucose-stimulated insulin secretion in the presence of proinflammatory cytokines and triggered gene expression pathways responsive to oxidative stress and cell death. Consistent with a role for 12/15-LOX in promoting oxidative stress, its chemical inhibition reduced production of reactive oxygen species in both mouse and human islets in vitro. In a streptozotocin-induced model of T1D in mice, ML351 prevented the development of diabetes, with coincident enhancement of nuclear Nrf2 in islet cells, reduced β-cell oxidative stress, and preservation of β-cell mass. In the nonobese diabetic mouse model of T1D, administration of ML351 during the prediabetic phase prevented dysglycemia, reduced β-cell oxidative stress, and increased the proportion of anti-inflammatory macrophages in insulitis. The data provide the first evidence to date that small molecules that target 12/15-LOX can prevent progression of β-cell dysfunction and glycemic deterioration in models of T1D.

This study tested the hypothesis that glucagon-like peptide 1 (GLP-1) therapies improve cardiac contractile function at rest and in response to adrenergic stimulation in obese swine after myocardial infarction. Obese Ossabaw swine were subjected to gradually developing regional coronary occlusion using an ameroid occluder placed around the left anterior descending coronary artery. Animals received subcutaneous injections of saline or liraglutide (0.005–0.015 mg/kg/day) for 30 days after ameroid placement. Cardiac performance was assessed at rest and in response to sympathomimetic challenge (dobutamine 0.3–10 μg/kg/min) using a left ventricular pressure/volume catheter. Liraglutide increased diastolic relaxation (dP/dt; Tau 1/2; Tau 1/e) during dobutamine stimulation (P < 0.01) despite having no influence on the magnitude of myocardial infarction. The slope of the end-systolic pressure volume relationship (i.e., contractility) increased with dobutamine after liraglutide (P < 0.001) but not saline administration (P = 0.63). Liraglutide enhanced the slope of the relationship between cardiac power and pressure volume area (i.e., cardiac efficiency) with dobutamine (P = 0.017). Hearts from animals treated with liraglutide demonstrated decreased β1-adrenoreceptor expression. These data support that GLP-1 agonism augments cardiac efficiency via attenuation of maladaptive sympathetic signaling in the setting of obesity and myocardial infarction.

The pancreatic islet is a complex micro-organ containing numerous cell types, including endocrine, immune, and endothelial cells. The communication of these systems is lost upon isolation of the islets, and therefore the pathogenesis of diabetes can only be fully understood by studying this organized, multicellular environment in vivo. We have developed several adaptable tools to create a versatile platform to interrogate β-cell function in vivo. Specifically, we developed β-cell-selective virally-encoded fluorescent protein biosensors that can be rapidly and easily introduced into any mouse. We then coupled the use of these biosensors with intravital microscopy, a powerful tool that can be used to collect cellular and subcellular data from living tissues. Together, these approaches allowed the observation of in vivo β-cell-specific ROS dynamics using the Grx1-roGFP2 biosensor and calcium signaling using the GcAMP6s biosensor. Next, we utilized abdominal imaging windows (AIW) to extend our in vivo observations beyond single-point terminal measurements to collect longitudinal physiological and biosensor data through repeated imaging of the same mice over time. This platform represents a significant advancement in our ability to study β-cell structure and signaling in vivo, and its portability for use in virtually any mouse model will enable meaningful studies of β-cell physiology in the endogenous islet niche.

Glucose-stimulated insulin secretion by pancreatic β-cells results in a high level of metabolic activity and ROS production. Excess ROS can cause cellular damage leading to apoptosis. We recently reported that the cytokine interleukin 6 (IL-6) stimulates autophagy and protects β-cells from ROS-induced damage and apoptosis via the autophagic degradation of an antioxidant response repressor, KEAP1. This results in increased protein levels of the master antioxidant factor, NRF2. IL-6 also stimulates early markers of mitophagy in a NRF2-dependent manner. Interestingly, these effects are associated with noncanonical translocation of NRF2 to the mitochondria. Here, we set out to validate these findings and determine how NRF2 mitochondrial translocation is regulated. Using fluorescence imaging of mt-keima, we demonstrate that IL-6 stimulates mitophagy. This occurs without effects on mitochondrial function as measured by the Seahorse MitoStress Test. We also find that IL-6 rapidly stimulates NRF2 mitochondrial translocation, an effect that does not occur in response to treatment with hydrogen peroxide or a chemical inhibitor of KEAP1. These data indicate that liberating NRF2 from KEAP1 alone does not lead to NRF2 mitochondrial translocation, suggesting that IL-6 regulates NRF2 localization by additional mechanisms. NRF2 has extensive post-translational modifications, some of which are associated with nuclear localization. Therefore, we hypothesized that IL-6 induces specific NRF2 post-translational modifications that promote mitochondrial translocation. Indeed, we found that IL-6 stimulates serine phosphorylation on NRF2. Based on our findings described here, we propose that selective modification of NRF2 regulates its distinct subcellular localization and that selective translocation determines the mechanism of NRF2-mediated antioxidant response that functions to reduce ROS and restore cellular homeostasis in pancreatic β-cells. Disclosure M. Marasco: Employee; Spouse/Partner; Eli Lilly and Company. A.M. Conteh: None. C. Muralidharan: None. E. Pfeifer: None. A.K. Linnemann: None. Funding National Institutes of Health (K01DK102492), (R03DK115990 to A.K.L.)

Pancreatic ductal adenocarcinomas (PDACs) are aggressive with frequent lymphatic spread. By analysis of data from The Cancer Genome Atlas, we determined that ~35% of PDACs have a pro-angiogenic gene signature. We now show that the same PDACs exhibit increased expression of lymphangiogenic genes and lymphatic endothelial cell (LEC) markers, and that LEC abundance in human PDACs correlates with endothelial cell microvessel density. Lymphangiogenic genes and LECs are also elevated in murine PDACs arising in the KRC (mutated Kras; deleted RB) and KIC (mutated Kras; deleted INK4a) genetic models. Moreover, pancreatic cancer cells (PCCs) derived from KRC tumors express and secrete high levels of lymphangiogenic factors, including the EGF receptor ligand, amphiregulin. Importantly, TGF-β1 increases lymphangiogenic genes and amphiregulin expression in KRC PCCs but not in murine PCCs that lack SMAD4, and combinatorial targeting of the TGF-β type I receptor (TβRI) with LY2157299 and EGFR/HER2 with lapatinib suppresses tumor growth and metastasis in a syngeneic orthotopic model, and attenuates tumor lymphangiogenesis and angiogenesis while reducing lymphangiogenic genes and amphiregulin and enhancing apoptosis. Therefore, this combination could be beneficial in PDACs with lymphangiogenic or angiogenic gene signatures.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, with an estimated global prevalence of 1 in 4 individuals. Aberrant transcriptional control of gene expression is central to the pathophysiology of metabolic diseases. However, the molecular mechanisms leading to gene dysregulation are not well understood. Histone modifications play important roles in the control of transcription. Acetylation of histone 3 at lysine 9 (H3K9ac) is associated with transcriptional activity and is implicated in transcript elongation by controlling RNA polymerase II (RNAPII) pause-release. Hence, changes in this histone modification may shed information on novel pathways linking transcription control and metabolic dysfunction. Here, we carried out genome-wide analysis of H3K9ac in the liver of mice fed a control or a high-fat diet (an animal model of NAFLD), and asked whether this histone mark associates with changes in gene expression. We found that over 70% of RNAPII peaks in promoter-proximal regions overlapped with H3K9ac, consistent with a role of H3K9ac in the regulation of transcription. When comparing high-fat with control diet, approximately 17% of the differentially expressed genes were associated with changes in H3K9ac in their promoters, showing a strong correlation between changes in H3K9ac signal and gene expression. Overall, our data indicate that in response to a high-fat diet, dysregulated gene expression of a subset of genes may be attributable to changes in transcription elongation driven by H3K9ac. Our results point at an added mechanism of gene regulation that may be important in the development of metabolic diseases.

Impaired function and apoptosis of insulin-secreting islet β-cells is central to disease progression in both type 1 and type 2 diabetes. Oxidative damage resulting from excess reactive oxygen species (ROS) is a central factor in β-cell dysfunction and death, but the dynamic nature of ROS accumulation and its depletion pose a problem for mechanistic studies in vivo. Biosensors, including the redox-sensitive GFP (roGFPs), coupled with intravital microscopy provide a sensitive and dynamic solution to this problem. Here, we utilize a virally-delivered roGFP2-containing human glutaredoxin-1 (Grx1-roGFP2) to selectively monitor β-cell ROS dynamics in vivo in response to toxic glucose analogs. We paired viral biosensor delivery with implanted abdominal imaging windows over the pancreas, thus allowing longitudinal measurements of β-cell ROS and islet area during and after streptozotocin (STZ) exposure. The studies presented here represent a robust experimental platform that could be readily adapted to various transgenic or physiological mouse models in conjunction with any number of available biosensors, and thus opens a vast realm of potential for discovery in islet biology in vivo.

It is well known that a chronic state of elevated reactive oxygen species (ROS) in pancreaticβ-cells impairs their ability to release insulin in response to elevated plasma glucose. Moreover, at its extreme, unmitigated ROS drives regulated cell death. This dysfunctional state of ROS buildup can result both from genetic predisposition and environmental factors such as obesity and overnutrition. Importantly, excessive ROS buildup may underlie metabolic pathologies such as type 2 diabetes mellitus. The ability to monitor ROS dynamics inβ-cells in situ and to manipulate it via genetic, pharmacological, and environmental means would accelerate the development of novel therapeutics that could abate this pathology. Currently, there is a lack of models with these attributes that are available to the field. In this study, we use a zebrafish model to demonstrate that ROS can be generated in aβ-cell-specific manner using a hybrid chemical genetic approach. Using a transgenic nitroreductase-expressing zebrafish line,Tg(ins:Flag-NTR)s950, treated with the prodrug metronidazole (MTZ), we found that ROS is rapidly and explicitly generated inβ-cells. Furthermore, the level of ROS generated was proportional to the dosage of prodrug added to the system. At high doses of MTZ, caspase 3 was rapidly cleaved,β-cells underwent regulated cell death, and macrophages were recruited to the islet to phagocytose the debris. Based on our findings, we propose a model for the mechanism of NTR/MTZ action in transgenic eukaryotic cells and demonstrate the robust utility of this system to model ROS-related disease pathology.

It is well known that a chronic state of elevated reactive oxygen species (ROS) in pancreatic β-cells impairs their ability to release insulin in response to elevated plasma glucose. Moreover, at its extreme, unmitigated ROS drives regulated cell death. This dysfunctional state of ROS buildup can result both from genetic predisposition and environmental factors such as obesity and overnutrition. Importantly, excessive ROS buildup may underlie metabolic pathologies such as type 2 diabetes mellitus. The ability to monitor ROS dynamics in β-cells in situ and to manipulate it via genetic, pharmacological, and environmental means would accelerate the development of novel therapeutics that could abate this pathology. Currently, there is a lack of models with these attributes that are available to the field. In this study, we use a zebrafish model to demonstrate that ROS can be generated in a β-cell-specific manner using a hybrid chemical genetic approach. Using a transgenic nitroreductase-expressing zebrafish line, Tg(ins:Flag-NTR)s950, treated with the prodrug metronidazole (MTZ), we found that ROS is rapidly and explicitly generated in β-cells. Furthermore, the level of ROS generated was proportional to the dosage of prodrug added to the system. At high doses of MTZ, caspase 3 was rapidly cleaved, β-cells underwent regulated cell death, and macrophages were recruited to the islet to phagocytose the debris. Based on our findings, we propose a model for the mechanism of NTR/MTZ action in transgenic eukaryotic cells and demonstrate the robust utility of this system to model ROS-related disease pathology.

Coronary perivascular adipose tissue is a naturally occurring adipose tissue depot that normally surrounds the major coronary arteries on the surface of the heart. Although originally thought to promote vascular health and integrity, there is a growing body of evidence to support that coronary perivascular adipose tissue displays a distinct phenotype relative to other adipose depots and is capable of producing local factors with the potential to augment coronary vascular tone, inflammation, and the initiation and progression of coronary artery disease. The purpose of the present review is to outline previous findings about the cardiovascular effects of coronary perivascular adipose tissue and the potential mechanisms by which adipose-derived factors may influence coronary vascular function and the progression of atherogenesis.

This study tested the hypothesis that obesity alters the cardiac response to ischemia/reperfusion and/or glucagon like peptide-1 (GLP-1) receptor activation, and that these differences are associated with alterations in the obese cardiac proteome and microRNA (miRNA) transcriptome. Ossabaw swine were fed normal chow or obesogenic diet for 6 months. Cardiac function was assessed at baseline, during a 30-minutes coronary occlusion, and during 2 hours of reperfusion in anesthetized swine treated with saline or exendin-4 for 24 hours. Cardiac biopsies were obtained from normal and ischemia/reperfusion territories. Fat-fed animals were heavier, and exhibited hyperinsulinemia, hyperglycemia, and hypertriglyceridemia. Plasma troponin-I concentration (index of myocardial injury) was increased following ischemia/reperfusion and decreased by exendin-4 treatment in both groups. Ischemia/reperfusion produced reductions in systolic pressure and stroke volume in lean swine. These indices were higher in obese hearts at baseline and relatively maintained throughout ischemia/reperfusion. Exendin-4 administration increased systolic pressure in lean swine but did not affect the blood pressure in obese swine. End-diastolic volume was reduced by exendin-4 following ischemia/reperfusion in obese swine. These divergent physiologic responses were associated with obesity-related differences in proteins related to myocardial structure/function (e.g. titin) and calcium handling (e.g. SERCA2a, histidine-rich Ca(2+) binding protein). Alterations in expression of cardiac miRs in obese hearts included miR-15, miR-27, miR-130, miR-181, and let-7. Taken together, these observations validate this discovery approach and reveal novel associations that suggest previously undiscovered mechanisms contributing to the effects of obesity on the heart and contributing to the actions of GLP-1 following ischemia/reperfusion.

e16585Background: There are multiple case reports of spontaneously regressing metastatic RCC and M patients but little information about the natural history of disease after regression has been doc...

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that has been shown to have hemodynamic and cardioprotective capacity in addition to its better characterized glucoregulatory actions. Because of this, emerging research has focused on the ability of GLP-1 based therapies to drive myocardial substrate selection, enhance cardiac performance and regulate heart rate, blood pressure and vascular tone. These studies have produced consistent and reproducible results amongst numerous laboratories. However, there are obvious disparities in findings obtained in small animal models versus those of higher mammals. This species dependent discrepancy calls to question, the translational value of individual findings. Moreover, few studies of GLP-1 mediated cardiovascular action have been performed in the presence of a pre-existing comorbidities (e.g. obesity/diabetes) which limits interpretation of the effectiveness of incretin-based therapies in the setting of disease. This review addresses cardiovascular and hemodynamic potential of GLP-1 based therapies with attention to species specific effects as well as the interaction between therapies and disease.

This study examined the cardiovascular effects of GLP-1 (7-36) or (9-36) on myocardial oxygen consumption, function and systemic hemodynamics in vivo during normal perfusion and during acute, regional myocardial ischemia. Lean Ossabaw swine received systemic infusions of saline vehicle or GLP-1 (7-36 or 9-36) at 1.5, 3.0, and 10.0 pmol/kg/min in sequence for 30 min at each dose, followed by ligation of the left circumflex artery during continued infusion at 10.0 pmol/kg/min. Systemic GLP-1 (9-36) had no effect on coronary flow, blood pressure, heart rate or indices of cardiac function before or during regional myocardial ischemia. Systemic GLP-1 (7-36) exerted no cardiometabolic or hemodynamic effects prior to ischemia. During ischemia, GLP-1 (7-36) increased cardiac output by approximately 2 L/min relative to vehicle-controls (p = 0.003). This response was not diminished by treatment with the non-depolarizing ganglionic blocker hexamethonium. Left ventricular pressure-volume loops measured during steady-state conditions with graded occlusion of the inferior vena cava to assess load-independent contractility revealed that GLP-1 (7-36) produced marked increases in end-diastolic volume (74 ± 1 to 92 ± 5 ml; p = 0.03) and volume axis intercept (8 ± 2 to 26 ± 8; p = 0.05), without any change in the slope of the end-systolic pressure-volume relationship vs. vehicle during regional ischemia. GLP-1 (9-36) produced no changes in any of these parameters compared to vehicle. These findings indicate that short-term systemic treatment with GLP-1 (7-36) but not GLP-1 (9-36) significantly augments cardiac output during regional myocardial ischemia, via increases in ventricular preload without changes in cardiac inotropy.

This study investigated the cardiovascular effects of the glucagon-like peptide-1 (GLP-1) receptor agonist Exendin-4 in lean vs. obese hearts. Left ventricular pressure-volume relations were obtained following 24 hr intravenous infusion of saline-vehicle or Exendin-4 (30 fmol/kg/min) in lean (69 ± 4 kg) and obese (105 ± 2 kg) Ossabaw swine with metabolic syndrome (MetS). Measurements were obtained at rest, following 30 min occlusion of the left circumflex coronary artery, and following 2 hours of reperfusion. Baseline coronary flow, heart rate, and stroke volume were not different between vehicle-treated lean and MetS swine. Exendin-4 had no effect on end diastolic volume or stroke volume in lean or MetS swine. However, cardiac output and blood pressure were significantly increased relative to end-diastolic filling volumes by Exendin-4 administration in lean swine (P

This study examined the cardiovascular effects of GLP-1 (7-36) vs. (9-36) in normal and ischemic hearts. Swine received systemic infusions of GLP-1 (7-36 or 9-36) at 1.5, 3.0, and 10.0 pmol/kg/min in sequence for 30 min at each dose. Subsequent to this dose response, vehicle or GLP-1 infusions were maintained at 10.0 pmol/kg/min and the left circumflex artery was ligated to induce regional ischemia. GLP-1 (9-36) had no effect on coronary flow, blood pressure, heart rate or indices of cardiac function before or during regional myocardial ischemia. GLP-1 (7-36) dose-dependently increased myocardial glucose uptake (1.3 + 0.8 to 5.5 + 1.9 μmol/min/g; p=0.05), but exerted no other cardiovascular effects prior to ischemia. During ischemia, GLP-1 (7-36) increased cardiac output by 2.4 L/min relative to vehicle-controls (p=0.003). Left ventricular pressure-volume loops measured during graded IVC occlusion revealed marked increases in end diastolic volume (74 + 1 to 92 + 5 mL;p=0.03) and calculated ventricular vol...

Exposure to herbicide-treated lawns has been associated with significantly higher bladder cancer risk in dogs. This work was performed to further characterize lawn chemical exposures in dogs, and to determine environmental factors associated with chemical residence time on grass. In addition to concern for canine health, a strong justification for the work was that dogs may serve as sentinels for potentially harmful environmental exposures in humans. Experimentally, herbicides [2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxypropionic acid (MCPP), dicamba] were applied to grass plots under different conditions (e.g., green, dry brown, wet, and recently mowed grass). Chemicals in dislodgeable residues were measured by LC-MS at 0.17, 1, 24, 48, 72 h post treatment. In a separate study, 2,4-D, MCPP, and dithiopyr concentrations were measured in the urine of dogs and in dislodgeable grass residues in households that applied or did not apply chemicals in the preceding 48 h. Chemicals were measured at 0, 24, and 48 h post application in treated households and at time 0 in untreated control households. Residence times of 2,4-D, MCPP, and dicamba were significantly prolonged (P