Machine and deep learning models for accurate detection of ischemia and scar with myocardial blood flow positron emission tomography imaging.

BACKGROUND: Quantification of myocardial blood flow (MBF) is used for the noninvasive diagnosis of patients with coronary artery disease (CAD). This study compared traditional statistics, machine learning, and deep learning techniques in their ability to diagnose disease using only the rest and stress MBF values. METHODS: This study included 3245 rest and stress rubidium-82 positron emission tomography (PET) studies and matching diagnostic labels from perfusion reports. Standard logistic regression, lasso logistic regression, support vector machine, random forest, multilayer perceptron, and dense U-Net were compared for per-patient detection and per-vessel localization of scars and ischemia. RESULTS: Receiver-operator characteristic area under the curve (AUC) of machine learning models was significantly higher than those of traditional statistics models for per-patient detection of disease (0.92-0.95 vs. 0.87) but not for per-vessel localization of ischemia or scar. Random forest showed the highest AUC = 0.95 among the different models compared. On the final hold-out set for generalizability, random forest showed an AUC of 0.92 for detection and 0.89 for localization of perfusion abnormalities. CONCLUSIONS: For per-vessel localization, simple models trained on segmental data performed similarly to a convolutional neural network trained on polar-map data, highlighting the need to justify the use of complex predictive algorithms through comparison with simpler methods.

ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression.

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1's role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

The SSBP3 co-regulator is required for glucose homeostasis, pancreatic islet architecture, and beta-cell identity.

OBJECTIVE: Transcriptional complex activity drives the development and function of pancreatic islet cells to allow for proper glucose regulation. Prior studies from our lab and others highlighted that the LIM-homeodomain transcription factor (TF), Islet-1 (Isl1), and its interacting co-regulator, Ldb1, are vital effectors of developing and adult ß-cells. We further found that a member of the Single Stranded DNA-Binding Protein (SSBP) co-regulator family, SSBP3, interacts with Isl1 and Ldb1 in ß-cells and primary islets (mouse and human) to impact ß-cell target genes MafA and Glp1R in vitro. Members of the SSBP family stabilize TF complexes by binding directly to Ldb1 and protecting the complex from ubiquitin-mediated turnover. In this study, we hypothesized that SSBP3 has critical roles in pancreatic islet cell function in vivo, similar to the Isl1::Ldb1 complex. METHODS: We first developed a novel SSBP3 LoxP allele mouse line, where Cre-mediated recombination imparts a predicted early protein termination. We bred this mouse with constitutive Cre lines (Pdx1- and Pax6-driven) to recombine SSBP3 in the developing pancreas and islet (SSBP3ΔPanc and SSBP3ΔIslet), respectively. We assessed glucose tolerance and used immunofluorescence to detect changes in islet cell abundance and markers of ß-cell identity and function. Using an inducible Cre system, we also deleted SSBP3 in the adult ß-cell, a model termed SSBP3Δß-cell. We measured glucose tolerance as well as glucose-stimulated insulin secretion (GSIS), both in vivo and in isolated islets in vitro. Using islets from control and SSBP3Δß-cell we conducted RNA-Seq and compared our results to published datasets for similar ß-cell specific Ldb1 and Isl1 knockouts to identify commonly regulated target genes. RESULTS: SSBP3ΔPanc and SSBP3ΔIslet neonates present with hyperglycemia. SSBP3ΔIslet mice are glucose intolerant by P21 and exhibit a reduction of ß-cell maturity markers MafA, Pdx1, and UCN3. We observe disruptions in islet cell architecture with an increase in glucagon+ α-cells and ghrelin+ ε-cells at P10. Inducible loss of ß-cell SSBP3 in SSBP3Δß-cell causes hyperglycemia, glucose intolerance, and reduced GSIS. Transcriptomic analysis of 14-week-old SSBP3Δß-cell islets revealed a decrease in ß-cell function gene expression (Ins, MafA, Ucn3), increased stress and dedifferentiation markers (Neurogenin-3, Aldh1a3, Gastrin), and shared differentially expressed genes between SSBP3, Ldb1, and Isl1 in adult ß-cells. CONCLUSIONS: SSBP3 drives proper islet identity and function, where its loss causes altered islet-cell abundance and glucose homeostasis. ß-Cell SSBP3 is required for GSIS and glucose homeostasis, at least partially through shared regulation of Ldb1 and Isl1 target genes.

The Ldb1 transcriptional co-regulator is required for establishment and maintenance of the pancreatic endocrine lineage.

Pancreatic islet cell development is regulated by transcription factors (TFs) that mediate embryonic progenitor differentiation toward mature endocrine cells. Prior studies from our lab and others showed that the islet-enriched TF, Islet-1 (Isl1), interacts with the broadly-expressed transcriptional co-regulator, Ldb1, to regulate islet cell maturation and postnhyperatal function (by embryonic day (E)18.5). However, Ldb1 is expressed in the developing pancreas prior to Isl1 expression, notably in multipotent progenitor cells (MPCs) marked by Pdx1 and endocrine progenitors (EPs) expressing Neurogenin-3 (Ngn3). MPCs give rise to the endocrine and exocrine pancreas, while Ngn3+ EPs specify pancreatic islet endocrine cells. We hypothesized that Ldb1 is required for progenitor identity in MPC and EP populations during development to impact islet appearance and function. To test this, we generated a whole-pancreas Ldb1 knockout, termed Ldb1ΔPanc , and observed severe developmental and postnatal pancreas defects including disorganized progenitor pools, a significant reduction of Ngn3-expressing EPs, Pdx1HI ß-cells, and early hormone+ cells. Ldb1ΔPanc neonates presented with severe hyperglycemia, hypoinsulinemia, and drastically reduced hormone expression in islets, yet no change in total pancreas mass. This supports the endocrine-specific actions of Ldb1. Considering this, we also developed an endocrine-enriched model of Ldb1 loss, termed Ldb1ΔEndo . We observed similar dysglycemia in this model, as well as a loss of islet identity markers. Through in vitro and in vivo chromatin immunoprecipitation experiments, we found that Ldb1 occupies key Pdx1 and Ngn3 promoter domains. Our findings provide insight into novel regulation of endocrine cell differentiation that may be vital toward improving cell-based diabetes therapies.

Islet transplantation into brown adipose tissue can delay immune rejection.

Type 1 diabetes is an autoimmune disease characterized by insulin-producing ß cell destruction. Although islet transplantation restores euglycemia and improves patient outcomes, an ideal transplant site remains elusive. Brown adipose tissue (BAT) has a highly vascularized and antiinflammatory microenvironment. Because these tissue features can promote islet graft survival, we hypothesized that islets transplanted into BAT will maintain islet graft and BAT function while delaying immune-mediated rejection. We transplanted syngeneic and allogeneic islets into BAT or under the kidney capsule of streptozotocin-induced diabetic NOD.Rag and NOD mice to investigate islet graft function, BAT function, metabolism, and immune-mediated rejection. Islet grafts within BAT restored euglycemia similarly to kidney capsule controls. Islets transplanted in BAT maintained expression of islet hormones and transcription factors and were vascularized. Compared with those in kidney capsule and euglycemic mock-surgery controls, no differences in glucose or insulin tolerance, thermogenic regulation, or energy expenditure were observed with islet grafts in BAT. Immune profiling of BAT revealed enriched antiinflammatory macrophages and T cells. Compared with the kidney capsule control, there were significant delays in autoimmune and allograft rejection of islets transplanted in BAT, possibly due to increased antiinflammatory immune populations. Our data support BAT as an alternative islet transplant site that may improve graft survival.

LDB1-mediated transcriptional complexes are sensitive to islet stress.

Excess nutrients and proinflammatory cytokines impart stresses on pancreatic islet ß-cells that, if unchecked, can lead to cellular dysfunction and/or death. Among these stress-induced effects is loss of key ß-cell transcriptional regulator mRNA and protein levels required for ß-cell function. Previously, our lab and others reported that LIM-domain complexes comprised the LDB1 transcriptional co-regulator and Islet-1 (ISL1) transcription factor are required for islet ß-cell development, maturation, and function. The LDB1:ISL1 complex directly occupies and regulates key ß-cell genes, including MafA, Pdx1, and Slc2a2, to maintain ß-cell identity and function. Given the importance of LDB1:ISL1 complexes, we hypothesized that LDB1 and/or ISL1 levels, like other transcriptional regulators, are sensitive to ß-cell nutrient and cytokine stresses, likely contributing to ß-cell (dys)function under various stimuli. We tested this by treating ß-cell lines or primary mouse islets with elevating glucose concentrations, palmitate, or a cytokine cocktail of IL-1ß, TNFα, and IFNγ. We indeed observed that LDB1 mRNA and/or protein levels were reduced upon palmitate and cytokine (cocktail or singly) incubation. Conversely, acute high glucose treatment of ß-cells did not impair LDB1 or ISL1 levels, but increased LDB1:ISL1 interactions. These observations suggest that LDB1:ISL1 complex formation is sensitive to ß-cell stresses and that targeting and/or stabilizing this complex may rescue lost ß-cell gene expression to preserve cellular function.

Partners in Crime: Beta-Cells and Autoimmune Responses Complicit in Type 1 Diabetes Pathogenesis.

Type 1 diabetes (T1D) is an autoimmune disease characterized by autoreactive T cell-mediated destruction of insulin-producing pancreatic beta-cells. Loss of beta-cells leads to insulin insufficiency and hyperglycemia, with patients eventually requiring lifelong insulin therapy to maintain normal glycemic control. Since T1D has been historically defined as a disease of immune system dysregulation, there has been little focus on the state and response of beta-cells and how they may also contribute to their own demise. Major hurdles to identifying a cure for T1D include a limited understanding of disease etiology and how functional and transcriptional beta-cell heterogeneity may be involved in disease progression. Recent studies indicate that the beta-cell response is not simply a passive aspect of T1D pathogenesis, but rather an interplay between the beta-cell and the immune system actively contributing to disease. Here, we comprehensively review the current literature describing beta-cell vulnerability, heterogeneity, and contributions to pathophysiology of T1D, how these responses are influenced by autoimmunity, and describe pathways that can potentially be exploited to delay T1D.

Xenotransplantation of tannic acid-encapsulated neonatal porcine islets decreases proinflammatory innate immune responses.

BACKGROUND: Islet transplantation with neonatal porcine islets (NPIs) is a promising treatment for type 1 diabetes (T1D), but immune rejection poses a major hurdle for clinical use. Innate immune-derived reactive oxygen species (ROS) synthesis can facilitate islet xenograft destruction and enhance adaptive immune responses. METHODS: To suppress ROS-mediated xenograft destruction, we utilized nanothin encapsulation materials composed of multilayers of tannic acid (TA), an antioxidant, and a neutral polymer, poly(N-vinylpyrrolidone) (PVPON). We hypothesized that (PVPON/TA)-encapsulated NPIs will maintain euglycemia and dampen proinflammatory innate immune responses following xenotransplantation. RESULTS: (PVPON/TA)-encapsulated NPIs were viable and glucose-responsive similar to non-encapsulated NPIs. Transplantation of (PVPON/TA)-encapsulated NPIs into hyperglycemic C57BL/6.Rag or NOD.Rag mice restored euglycemia, exhibited glucose tolerance, and maintained islet-specific transcription factor levels similar to non-encapsulated NPIs. Gene expression analysis of (PVPON/TA)-encapsulated grafts post-transplantation displayed reduced proinflammatory Ccl5, Cxcl10, Tnf, and Stat1 while enhancing alternatively activated macrophage Retnla, Arg1, and Stat6 mRNA accumulation compared with controls. Flow cytometry analysis demonstrated significantly reduced innate immune infiltration, MHC-II, co-stimulatory molecule, and TNF expression with concomitant increases in arginase-1+ macrophages and dendritic cells. Similar alterations in immune responses were observed following xenotransplantation into immunocompetent NOD mice. CONCLUSION: Our data suggest that (PVPON/TA) encapsulation of NPIs is an effective strategy to decrease inflammatory innate immune signals involved in NPI xenograft responses through STAT1/6 modulation without compromising islet function.

The transcriptional co-regulator LDB1 is required for brown adipose function.

OBJECTIVE: Brown adipose tissue (BAT) is critical for thermogenesis and glucose/lipid homeostasis. Exploiting the energy uncoupling capacity of BAT may reveal targets for obesity therapies. This exploitation requires a greater understanding of the transcriptional mechanisms underlying BAT function. One potential regulator of BAT is the transcriptional co-regulator LIM domain-binding protein 1 (LDB1), which acts as a dimerized scaffold, allowing for the assembly of transcriptional complexes. Utilizing a global LDB1 heterozygous mouse model, we recently reported that LDB1 might have novel roles in regulating BAT function. However, direct evidence for the LDB1 regulation of BAT thermogenesis and substrate utilization has not been elucidated. We hypothesize that brown adipocyte-expressed LDB1 is required for BAT function. METHODS: LDB1-deficient primary cells and brown adipocyte cell lines were assessed via qRT-PCR and western blotting for altered mRNA and protein levels to define the brown adipose-specific roles. We conducted chromatin immunoprecipitation with primary BAT tissue and immortalized cell lines. Potential transcriptional partners of LDB1 were revealed by conducting LIM factor surveys via qRT-PCR in mouse and human brown adipocytes. We developed a Ucp1-Cre-driven LDB1-deficiency mouse model, termed Ldb1ΔBAT, to test LDB1 function in vivo. Glucose tolerance and uptake were assessed at thermoneutrality via intraperitoneal glucose challenge and glucose tracer studies. Insulin tolerance was measured at thermoneutrality and after stimulation with cold or the administration of the ß3-adrenergic receptor (ß3-AR) agonist CL316,243. Additionally, we analyzed plasma insulin via ELISA and insulin signaling via western blotting. Lipid metabolism was evaluated via BAT weight, histology, lipid droplet morphometry, and the examination of lipid-associated mRNA. Finally, energy expenditure and cold tolerance were evaluated via indirect calorimetry and cold challenges. RESULTS: Reducing Ldb1 in vitro and in vivo resulted in altered BAT-selective mRNA, including Ucp1, Elovl3, and Dio2. In addition, there was reduced Ucp1 induction in vitro. Impacts on gene expression may be due, in part, to LDB1 occupying Ucp1 upstream regulatory domains. We also identified BAT-expressed LIM-domain factors Lmo2, Lmo4, and Lhx8, which may partner with LDB1 to mediate activity in brown adipocytes. Additionally, we observed LDB1 enrichment in human brown adipose. In vivo analysis revealed LDB1 is required for whole-body glucose and insulin tolerance, in part through reduced glucose uptake into BAT. In Ldb1ΔBAT tissue, we found significant alterations in insulin-signaling effectors. An assessment of brown adipocyte morphology and lipid droplet size revealed larger and more unilocular brown adipocytes in Ldb1ΔBAT mice, particularly after a cold challenge. Alterations in lipid handling were further supported by reductions in mRNA associated with fatty acid oxidation and mitochondrial respiration. Finally, LDB1 is required for energy expenditure and cold tolerance in both male and female mice. CONCLUSIONS: Our findings support LDB1 as a regulator of BAT function. Furthermore, given LDB1 enrichment in human brown adipose, this co-regulator may have conserved roles in human BAT.

Glucagon receptor signaling regulates weight loss via central KLB receptor complexes.

Glucagon regulates glucose and lipid metabolism and promotes weight loss. Thus, therapeutics stimulating glucagon receptor (GCGR) signaling are promising for obesity treatment; however, the underlying mechanism(s) have yet to be fully elucidated. We previously identified that hepatic GCGR signaling increases circulating fibroblast growth factor 21 (FGF21), a potent regulator of energy balance. We reported that mice deficient for liver Fgf21 are partially resistant to GCGR-mediated weight loss, implicating FGF21 as a regulator of glucagon's weight loss effects. FGF21 signaling requires an obligate coreceptor (ß-Klotho, KLB), with expression limited to adipose tissue, liver, pancreas, and brain. We hypothesized that the GCGR-FGF21 system mediates weight loss through a central mechanism. Mice deficient for neuronal Klb exhibited a partial reduction in body weight with chronic GCGR agonism (via IUB288) compared with controls, supporting a role for central FGF21 signaling in GCGR-mediated weight loss. Substantiating these results, mice with central KLB inhibition via a pharmacological KLB antagonist, 1153, also displayed partial weight loss. Central KLB, however, is dispensable for GCGR-mediated improvements in plasma cholesterol and liver triglycerides. Together, these data suggest GCGR agonism mediates part of its weight loss properties through central KLB and has implications for future treatments of obesity and metabolic syndrome.

Glucagon-Receptor Signaling Reverses Hepatic Steatosis Independent of Leptin Receptor Expression.

Glucagon (GCG) is an essential regulator of glucose and lipid metabolism that also promotes weight loss. We have shown that glucagon-receptor (GCGR) signaling increases fatty acid oxidation (FAOx) in primary hepatocytes and reduces liver triglycerides in diet-induced obese (DIO) mice; however, the mechanisms underlying this aspect of GCG biology remains unclear. Investigation of hepatic GCGR targets elucidated a potent and previously unknown induction of leptin receptor (Lepr) expression. Liver leptin signaling is known to increase FAOx and decrease liver triglycerides, similar to glucagon action. Therefore, we hypothesized that glucagon increases hepatic LEPR, which is necessary for glucagon-mediated reversal of hepatic steatosis. Eight-week-old control and liver-specific LEPR-deficient mice (LeprΔliver) were placed on a high-fat diet for 12 weeks and then treated with a selective GCGR agonist (IUB288) for 14 days. Liver triglycerides and gene expression were assessed in liver tissue homogenates. Administration of IUB288 in both lean and DIO mice increased hepatic Lepr isoforms a-e in acute (4 hours) and chronic (72 hours,16 days) (P < 0.05) settings. LeprΔliver mice displayed increased hepatic triglycerides on a chow diet alone (P < 0.05), which persisted in a DIO state (P < 0.001), with no differences in body weight or composition. Surprisingly, chronic administration of IUB288 in DIO control and LeprΔliver mice reduced liver triglycerides regardless of genotype (P < 0.05). Together, these data suggest that GCGR activation induces hepatic Lepr expression and, although hepatic glucagon and leptin signaling have similar liver lipid targets, these appear to be 2 distinct pathways.

LIM-domain transcription complexes interact with ring-finger ubiquitin ligases and thereby impact islet ß-cell function.

Diabetes is characterized by a loss of ß-cell mass, and a greater understanding of the transcriptional mechanisms governing ß-cell function is required for future therapies. Previously, we reported that a complex of the Islet-1 (Isl1) transcription factor and the co-regulator single-stranded DNA-binding protein 3 (SSBP3) regulates the genes necessary for ß-cell function, but few proteins are known to interact with this complex in ß-cells. To identify additional components, here we performed SSBP3 reverse-cross-linked immunoprecipitation (ReCLIP)- and MS-based experiments with mouse ß-cell extracts and compared the results with those from our previous Isl1 ReCLIP study. Our analysis identified the E3 ubiquitin ligases ring finger protein 20 (RNF20) and RNF40, factors that in nonpancreatic cells regulate transcription through imparting monoubiquitin marks on histone H2B (H2Bub1), a precursor to histone H3 lysine 4 trimethylation (H3K4me3). We hypothesized that RNF20 and RNF40 regulate similar genes as those regulated by Isl1 and SSBP3 and are important for ß-cell function. We observed that Rnf20 and Rnf40 depletion reduces ß-cell H2Bub1 marks and uncovered several target genes, including glucose transporter 2 (Glut2), MAF BZIP transcription factor A (MafA), and uncoupling protein 2 (Ucp2). Strikingly, we also observed that Isl1 and SSBP3 depletion reduces H2Bub1 and H3K4me3 marks, suggesting that they have epigenetic roles. We noted that the RNF complex is required for glucose-stimulated insulin secretion and normal mitochondrial reactive oxygen species levels. These findings indicate that RNF20 and RNF40 regulate ß-cell gene expression and insulin secretion and establish a link between Isl1 complexes and global cellular epigenetics.

Prolactin Receptor Signaling Regulates a Pregnancy-Specific Transcriptional Program in Mouse Islets.

Pancreatic ß-cells undergo profound hyperplasia during pregnancy to maintain maternal euglycemia. Failure to reprogram ß-cells into a more replicative state has been found to underlie susceptibility to gestational diabetes mellitus (GDM). We recently identified a requirement for prolactin receptor (PRLR) signaling in the metabolic adaptations to pregnancy, where ß-cell-specific PRLR knockout (ßPRLRKO) mice exhibit a metabolic phenotype consistent with GDM. However, the underlying transcriptional program that is responsible for the PRLR-dependent metabolic adaptations during gestation remains incompletely understood. To identify PRLR signaling gene regulatory networks and target genes within ß-cells during pregnancy, we performed a transcriptomic analysis of pancreatic islets isolated from either ßPRLRKO mice or littermate controls in late gestation. Gene set enrichment analysis identified forkhead box protein M1 and polycomb repressor complex 2 subunits, Suz12 and enhancer of zeste homolog 2 (Ezh2), as novel candidate regulators of PRLR-dependent ß-cell adaptation. Gene ontology term pathway enrichment revealed both established and novel PRLR signaling target genes that together promote a state of increased cellular metabolism and/or proliferation. In contrast to the requirement for ß-cell PRLR signaling in maintaining euglycemia during pregnancy, PRLR target genes were not induced following high-fat diet feeding. Collectively, the current study expands our understanding of which transcriptional regulators and networks mediate gene expression required for islet adaptation during pregnancy. The current work also supports the presence of pregnancy-specific adaptive mechanisms distinct from those activated by nutritional stress.

Patient body motion correction for dynamic cardiac PET-CT by attenuation-emission alignment according to projection consistency conditions.

INTRODUCTION: Patient body motion is known to cause large deviations in the determination of myocardial blood flow (MBF) with errors exceeding 300%. Accurate correction for patient whole-body motion is still a largely unsolved problem in cardiac positron emission tomography (PET) imaging. OBJECTIVE: This study evaluated the efficacy of using Natterer's formulation of the Helgason-Ludwig consistency conditions on the two-dimensional Radon transform to align computed tomography to PET projection data in multiple time frames of a dynamic sequence for the purpose of frame-by-frame correction of rigid whole-body motion. METHODS: The correction algorithm was evaluated with digital NCAT phantoms using realistic noise added by the analytical simulator. Count rates used in the simulation were derived from clinical patient data. In addition, a proof of concept test using measured data with a cardiac torso phantom was conducted. RESULTS: Motion correction resulted in significant improvement in the accuracy of MBF estimates, especially for high count-rate acquisitions. Maximum errors for 2 cm of motion dropped from 325% to 25% and from 250% to 25% using global and regional partial-volume correction, respectively. Median MBF errors dropped from 33% to 4.5% and 27% to 3.8%, respectively. Importantly, the correction algorithm performed equally well to compensate for body motion in both early and late time frames. CONCLUSION: Cardiac PET-CT data used for attenuation correction (CTAC) alignment using projection consistency conditions was effective for reducing errors in MBF measurements due to simulated patient motion, and can be integrated into the image reconstruction workflow.

The islet-expressed Lhx1 transcription factor interacts with Islet-1 and contributes to glucose homeostasis.

The LIM-homeodomain (LIM-HD) transcription factor Islet-1 (Isl1) interacts with the LIM domain-binding protein 1 (Ldb1) coregulator to control expression of key pancreatic ß-cell genes. However, Ldb1 also has Isl1-independent effects, supporting that another LIM-HD factor interacts with Ldb1 to impact ß-cell development and/or function. LIM homeobox 1 (Lhx1) is an Isl1-related LIM-HD transcription factor that appears to be expressed in the developing mouse pancreas and in adult islets. However, roles for this factor in the pancreas are unknown. This study aimed to determine Lhx1 interactions and elucidate gene regulatory and physiological roles in the pancreas. Co-immunoprecipitation using ß-cell extracts demonstrated an interaction between Lhx1 and Isl1, and thus we hypothesized that Lhx1 and Isl1 regulate similar target genes. To test this, we employed siRNA-mediated Lhx1 knockdown in ß-cell lines and discovered reduced Glp1R mRNA. Chromatin immunoprecipitation revealed Lhx1 occupancy at a domain also known to be occupied by Isl1 and Ldb1. Through development of a pancreas-wide knockout mouse model ( Lhx1∆Panc), we demonstrate that aged Lhx1∆Panc mice have elevated fasting blood glucose levels, altered intraperitoneal and oral glucose tolerance, and significantly upregulated glucagon, somatostatin, pancreatic polypeptide, MafB, and Arx islet mRNAs. Additionally, Lhx1∆Panc mice exhibit significantly reduced Glp1R, an mRNA encoding the insulinotropic receptor for glucagon-like peptide 1 along with a concomitant dampened Glp1 response and mild glucose intolerance in mice challenged with oral glucose. These data are the first to reveal that the Lhx1 transcription factor contributes to normal glucose homeostasis and Glp1 responses.

Kernel-Based Reconstruction of C-11-Hydroxyephedrine Cardiac PET Images of the Sympathetic Nervous System.

Image reconstruction for positron emission tomography (PET) can be challenging and the resulting image typically has high noise. The kernel-based reconstruction method [1], incorporates prior anatomic information in the reconstruction algorithm to reduce noise while preserving resolution. Prior information is incorporated in the reconstruction algorithm by means of spatial kernels originally used in machine learning. In this paper, the kernel-based method is used to reconstruct PET images of sympathetic innervation in the heart. The resulting images are compared with standard Ordered Subset Expectation Maximization (OSEM) reconstructed images qualitatively and quantitatively using data from 6 human subjects. The kernel-based method demonstrated superior SNR with preserved contrast and accuracy compared to OSEM.

Effects of Hypercapnia on Myocardial Blood Flow in Healthy Human Subjects.

Elevation of the end-tidal partial pressure of CO2 (PETco2) increases cerebral and myocardial blood flow (MBF), suggesting that it may be a suitable alternative to pharmacologic stress or exercise for myocardial perfusion imaging. The purpose of this study was to document the pharmacodynamics of CO2 for MBF using prospective end-tidal targeting to precisely control arterial Pco2 and PET to measure the outcome variable, MBF. Methods: Ten healthy men underwent serial 82Rb PET/CT imaging. Imaging was performed at rest and during 6-min hypercapnic plateaus (baseline; PETco2 at 50, 55, and 60 mm Hg; repeat of PETco2 at 60 mm Hg; and repeat of baseline). MBF was measured using 82Rb injected 3 min after the beginning of hypercapnia and a 1-tissue-compartment model with flow-dependent extraction correction. Results were compared with those obtained during an adenosine stress test (140 µg/kg/min). Results: Baseline PETco2 was 38.9 ± 0.8 (mean ± SD) mm Hg (range, 35-43 mm Hg). All PETco2 targets were sustained, with SDs of less than 1.5 mm Hg. Heart rate, systolic blood pressure, rate × pressure product, and respiratory frequency increased with progressive hypercapnia. MBF increased significantly at each level of hypercapnia to 1.92-fold over baseline (0.86 ± 0.24 vs. 0.45 ± 0.08 mL/min/g; P = 0.002) at a PETco2 of 60 mm Hg. MBF after the administration of adenosine was significantly greater than that with the maximal hypercapnic stimulus (2.00 vs. 0.86 mL/min/g; P < 0.0001). Conclusion: To our knowledge, this study is the first to assess the response of MBF to different levels of hypercapnia in healthy humans with PET. MBF increased with increasing levels of hypercapnia; MBF at a PETco2 of 60 mm Hg was double that at baseline.

Randomized Trial Comparing the Effects of Ticagrelor Versus Clopidogrel on Myocardial Perfusion in Patients With Coronary Artery Disease.

BACKGROUND: Ticagrelor is a P2Y12 receptor inhibitor used in acute coronary syndromes to reduce platelet activity and to decrease thrombus formation. Ticagrelor is associated with a reduction in mortality incremental to that observed with clopidogrel, potentially related to its non-antiplatelet effects. Evidence from animal models indicates that ticagrelor potentiates adenosine-induced myocardial blood flow (MBF) increases. We investigated MBF at rest and during adenosine-induced hyperemia in patients with stable coronary artery disease treated with ticagrelor versus clopidogrel. METHODS AND RESULTS: This randomized double-blinded crossover study included 22 patients who received therapeutic interventions of ticagrelor 90 mg orally twice a day for 10 days and clopidogrel 75 mg orally once a day for 10 days, with a washout period of at least 10 days between the treatments. Global and regional MBF and myocardial flow reserve were measured using rubidium 82 positron emission tomography/computed tomography at baseline and during intermediate- and high-dose adenosine. Global MBF was significantly greater with ticagrelor versus clopidogrel (1.28±0.55 versus 1.13±0.47 mL/min per gram, P=0.002) at intermediate-dose adenosine and not different at baseline (0.65±0.19 versus 0.60±0.15 mL/min per gram, P=0.084) and at high-dose adenosine (1.64±0.40 versus 1.61±0.19 mL/min per gram, P=0.53). In regions with impaired myocardial flow reserve (<2.5), MBF was greater with ticagrelor compared with clopidogrel during intermediate and high doses of adenosine (P<0.0001), whereas the differences were not significant at baseline. CONCLUSIONS: Ticagrelor potentiates global and regional adenosine-induced MBF increases in patients with stable coronary artery disease. This effect may contribute to the incremental mortality benefit compared with clopidogrel. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01894789.

Evidence for Loss in Identity, De-Differentiation, and Trans-Differentiation of Islet ß-Cells in Type 2 Diabetes.

The two main types of diabetes mellitus have distinct etiologies, yet a similar outcome: loss of islet ß-cell function that is solely responsible for the secretion of the insulin hormone to reduce elevated plasma glucose toward euglycemic levels. Type 1 diabetes (T1D) has traditionally been characterized by autoimmune-mediated ß-cell death leading to insulin-dependence, whereas type 2 diabetes (T2D) has hallmarks of peripheral insulin resistance, ß-cell dysfunction, and cell death. However, a growing body of evidence suggests that, especially during T2D, key components of ß-cell failure involves: (1) loss of cell identity, specifically proteins associated with mature cell function (e.g., insulin and transcription factors like MAFA, PDX1, and NKX6.1), as well as (2) de-differentiation, defined by regression to a progenitor or stem cell-like state. New technologies have allowed the field to compare islet cell characteristics from normal human donors to those under pathophysiological conditions by single cell RNA-Sequencing and through epigenetic analysis. This has revealed a remarkable level of heterogeneity among histologically defined "insulin-positive" ß-cells. These results not only suggest that these ß-cell subsets have different responses to insulin secretagogues, but that defining their unique gene expression and epigenetic modification profiles will offer opportunities to develop cellular therapeutics to enrich/maintain certain subsets for correcting pathological glucose levels. In this review, we will summarize the recent literature describing how ß-cell heterogeneity and plasticity may be influenced in T2D, and various possible avenues of therapeutic intervention.

Islet encapsulation with polyphenol coatings decreases pro-inflammatory chemokine synthesis and T cell trafficking.

Type 1 Diabetes (T1D) is a chronic pro-inflammatory autoimmune disease consisting of islet-infiltrating leukocytes involved in pancreatic ß-cell lysis. One promising treatment for T1D is islet transplantation; however, clinical application is constrained due to limited islet availability, adverse effects of immunosuppressants, and declining graft survival. Islet encapsulation may provide an immunoprotective barrier to preserve islet function and prevent immune-mediated rejection after transplantation. We previously demonstrated that a novel cytoprotective nanothin multilayer coating for islet encapsulation consisting of tannic acid (TA), an immunomodulatory antioxidant, and poly(N-vinylpyrrolidone) (PVPON), was efficacious in dampening in vitro immune responses involved in transplant rejection and preserving in vitro islet function. However, the ability of (PVPON/TA) to maintain islet function in vivo and reverse diabetes has not been tested. Recent evidence has demonstrated that modulation of redox status can affect pro-inflammatory immune responses. Therefore, we hypothesized that transplanted (PVPON/TA)-encapsulated islets can restore euglycemia to diabetic mice and provide an immunoprotective barrier. Our results demonstrate that (PVPON/TA) nanothin coatings can significantly decrease in vitro chemokine synthesis and diabetogenic T cell migration. Importantly, (PVPON/TA)-encapsulated islets restored euglycemia after transplantation into diabetic mice. Our results demonstrate that (PVPON/TA)-encapsulated islets may suppress immune responses and enhance islet allograft acceptance in patients with T1D.