bluuebunny/crossref_metadata_2025_split huggingface dataset:
CRISPR-Engineered Human Brown-Like Adipocytes Promotes Metabolic Homeostasis through Activation of Endogenous Brown Fat
Authors: WANG CHIH-HAO , LUNDH MORTEN , KRISZT ROKUS , HUANG TIAN LIAN , LEIRIA LUIZ O. , SHAMSI FARNAZ , LYNES MATT , DARCY JUSTIN , EMANUELLI BRICE , KIEBISH MICHAEL , TSENG YU-HUA 
Published: June 2018

Brown and brown-like beige adipocytes dissipate energy for thermogenesis and have been proposed to combat obesity and metabolic disorders. Uncoupling protein 1 (UCP1) is uniquely expressed in brown and beige adipocytes, and facilitates fuel utilization and energy expenditure. Ectopic UCP1 overexpression improves obesity and insulin resistance in mice. Recently, we utilized the CRISPR/Cas9 synergistic activation mediator and specific gRNAs to boost endogenous UCP1 expression in human white adipocytes. The CRISPR/Cas9 engineered human brown fat-like (referred to as HUMBLE, hereafter) cells had acquired human brown fat features when comparing bona fide human brown adipocytes derived from the same individual. Obese mice receiving a transplantation of HUMBLE cells showed improved glucose tolerance and insulin sensitivity, as well as increased energy expenditure. This was mediated, at least in part, via activation of endogenous brown fat, which showed an increase in heat production and glucose uptake in mice receiving HUMBLE cells. Co-culture with HUMBLE cells or treatment of conditional medium from HUMBLE cells promoted glucose uptake and thermogenesis in brown adipocytes. Metabolomic analysis identified an increased ratio of arginine and asymmetrical dimethylarginine (ADMA) in the sera of mice receiving HUMBLE cell transplantation. Arginine is known to promote nitric oxide biosynthesis, which can activate thermogenic function of brown fat. Consistently, the level of nitric oxide was increased in the endogenous brown fat of mice receiving HUMBLE cells. Inhibition of nitric oxide synthase abolished the HUMBLE cells-mediated activation of brown adipocytes in vitro and in vivo.
In summary, CRISPR/Cas9-mediated activation of endogenous UCP1 expression in human white adipocytes creates brown fat-like features, and activates endogenous brown fat, potentially through arginine-related metabolites to improve the systemic glucose utilization.

Disclosure
C. Wang: None. M. Lundh: None. R. Kriszt: Employee; Self; QuintilesIMS. T. Huang: None. L.O. Leiria: None. F. Shamsi: None. M. Lynes: None. J. Darcy: None. B. Emanuelli: None. M. Kiebish: None. Y. Tseng: Other Relationship; Self; Chugai Pharmaceutical Co., Ltd.. Research Support; Self; MedImmune.
Targeting Intestinal Macrophages as a Potential Therapeutic Option in Obesity
Authors: ROHM THERESA V. , ALASFOOR SHEFAA , BOSCH ANGELA J. , CAVELTI-WEDER CLAUDIA 
Published: June 2018

Background: Chronic inflammation such as systemic or tissue inflammation has been well established in metabolic disease. Although macrophages thereby play a key role, not much is known about the initiation of inflammation. As the gut makes up the largest macrophage reservoir of the body and gastrointestinal changes occur in metabolic disease (altered gut microbiota, increased endotoxin and cytokines), the aim of our study was to assess the role of intestinal macrophages (iMϕs) in obesity.
Research Design and Method: IMϕs were isolated from the colon of C57BL/6, germ-free or CCR2-/- mice fed a high fat diet (HFD) or control diet up to three months and characterized by flow cytometry as CCR2+ (“inflammatory”) or CCR2- (resident, anti-inflammatory) subpopulations. For dose-dependent iMϕ depletion, HFD-fed mice were orally treated with 50, 100 or 200 µg/g CSF-1R inhibitor (BLZ945) or its vehicle for up to ten weeks.
Results: We found that within one week and up to three months of HFD, inflammatory iMϕs increase, preceding adipose tissue inflammation. Two mouse models protected from metabolic disease - germ-free and CCR2-/- mice - exhibited ≥10-fold lower inflammatory iMϕ numbers when compared to WT mice, suggesting that a threshold of iMϕs is required to elicit metabolic disease. Based on this finding, we depleted macrophages by increasing doses of CSF-1R inhibitor and observed a gradual iMϕ-reduction, which correlated with a dose-dependent improvement in fasting glucose, glucose tolerance and insulin levels.
Conclusion: HFD-induced obesity is associated with an increase in inflammatory iMϕs, while mouse models protected from metabolic disease have lower numbers. Accordingly, dose-dependent depletion of iMϕs is accompanied by gradual improvements in glucose metabolism. This suggests that the number of iMϕs is involved in initiation of metabolic disease and thus could serve as a potential therapeutic target in obesity.

Disclosure
T.V. Rohm: None. S. AlAsfoor: None. A.J. Bosch: None. C. Cavelti-Weder: None.
Liraglutide Suppresses Atherosclerosis via AMP-Activated Protein Kinase Dependent and Independent Mechanisms in Diabetic Apolipoprotein Enull Mice
Authors: KOSHIBU MASAKAZU , MORI YUSAKU , KUSHIMA HIDEKI , HIROMURA MUNENORI , KOHASHI KYOKO , TERASAKI MICHISHIGE , HIRANO TSUTOMU 
Published: June 2018

Background: Glucagon like peptide 1 receptor agonists (GLP-1RAs) have anti-atherosclerotic properties, possibly through activation of AMP-activated protein kinase (AMPK) in the vasculature. However, their relationship remains largely unknown.
Methods: Streptozotocin-induced diabetic apolipoproteinE-null mice (male, 20 w) were randomly assigned to six treatment groups: saline (control, C), liraglutide at low or high doses (L- and H-Lira at 17 and 107 nmol/kg/d, respectively), AMPK inhibitor dorsomorphin (AMPKI, 25 mg/kg/d), AMPKI+L-Lira, or AMPKI+H-Lira.
Results: First, we confirmed that AMPKI inhibited H-Lira-induced AMPK phosphorylation in the aorta (1.5-fold). After 4 w, biological parameters were similar, except for HbA1c levels (C 9.8, L-Lira 8.6, H-Lira 8.4%, p&lt;0.05). Both L- and H-Lira suppressed intraplaque macrophage accumulation at the aortic sinus by 60%. H-Lira also suppressed aortic surface plaque area by 40%, which was not significant in L-Lira (25%). In AMPKI-treated mice, biological parameters were similar. AMPKI completely eliminated L-Lira-induced anti-atherogenic effects but did not attenuate H-Lira-induced suppression of atherosclerotic area (38%) and intraplaque macrophage accumulation (63%), suggesting that AMPK mechanisms are L-Lira-dependent and H-Lira-independent. In cultured human vascular endothelial cells, Lira (100 nM) reduced TNF-induced inflammatory cytokine and adhesion molecule expression by 60 to 80%, which was retained at more than 50% after siRNA-knockdown of AMPK (p&lt;0.05). In cultured human monocytic cells (U937), Lira suppressed LPS-induced inflammatory cytokine expression by 30% (p&lt;0.05), which was completely reversed by AMPKI.
Conclusions: Lira suppressed atherosclerosis via AMPK-dependent and -independent mechanisms; AMPK dependency is altered by GLP-1RA dose and cell type.

Disclosure
M. Koshibu: None. Y. Mori: None. H. Kushima: None. M. Hiromura: None. K. Kohashi: None. M. Terasaki: None. T. Hirano: Speaker's Bureau; Self; Novo Nordisk Inc., AstraZeneca.
Differential Effects of the Insulin-Only and Bihormonal Configurations of the Bionic Pancreas on Mean Glucose and Hypoglycemia during the Daytime and Nighttime
Authors: SHERWOOD JORDAN , BALLIRO COURTNEY A. , JAFRI RABAB Z. , EL-KHATIB FIRAS , MAHENO MICHELE , HILLARD MALLORY A. , O'DONOVAN ALEXANDER J. , SELAGAMSETTY RAJENDRANATH , ZHENG HUI , DAMIANO EDWARD , RUSSELL STEVEN J. 
Published: June 2018

The bihormonal bionic pancreas (BP) reduced both mean CGM glucose (CGMG) and time in the hypoglycemic range relative to usual care in outpatient studies. We recently performed an outpatient study in which we compared insulin-only (IOBP) and bihormonal (BHBP) configurations of the BP with usual care (UC; conventional or sensor-augmented insulin pump therapy) both with and without remote monitoring. We previously reported that monitoring did not affect the mean CGMG or hypoglycemia with either configuration of the BP. The current analysis focuses on the data collected from arms without remote monitoring (how the BP is eventually meant to be used) to determine how the effects of the BP differ between the day and night. As shown in the Table, both the IOBP (with glucose target of 110 mg/dl) and BHBP (with glucose target of 100 mg/dl) significantly reduced mean CGMG vs. UC during both the daytime and nighttime. In contrast, only the BHBP significantly reduced the percent of time spent with CGMG &lt;60 mg/dl vs. UC. The BHBP similarly reduced percent of time &lt;60 mg/dl vs. IOBP. The significant reductions in hypogycemia associated with the BHBP were driven primarily by reductions in hypoglycemia at night.

Disclosure
J. Sherwood: None. C.A. Balliro: None. R.Z. Jafri: None. F. El-Khatib: Stock/Shareholder; Self; Beta Bionics. Employee; Self; Beta Bionics. M. Maheno: None. M.A. Hillard: None. A.J. O'Donovan: None. R. Selagamsetty: None. H. Zheng: None. E. Damiano: Other Relationship; Self; Beta Bionics. S.J. Russell: Other Relationship; Self; Beta Bionics, Novo Nordisk Inc.. Advisory Panel; Self; Companion Medical, Tandem Diabetes Care, Inc., Unomedical a/s. Research Support; Self; Beta Bionics, Zealand Pharma A/S, MITRE Corporation.
NOX, NOX, Are You Here? The Emerging Role of NOX5 in Diabetic Neuropathy
Authors: EID STEPHANIE , HAYES JOHN M. , GUO KAI , PACUT CRYSTAL , MENDELSON FAYE , HUR JUNGUK , FELDMAN EVA L. 
Published: June 2018

Diabetic neuropathy (DN) is one of the most common complications of diabetes worldwide, affecting up to 60% of all diabetic patients. Although the pathogenesis of DN is poorly understood, it is widely accepted that reactive oxygen species (ROS) mediate in part the cellular and molecular injury observed in DN. NADPH oxidase (Nox) enzymes generate ROS and of the 7 isoforms, NOX5 is present only in man. In this study, we aimed to investigate a role for NOX5 in DN by identifying the cellular localization, methylation status and expression levels of NOX5 in cutaneous nerve fibers and sural nerve biopsies of diabetic patients. Cellular localization of NOX5, myelin basic protein (MBP) and protein gene product (PGP) 9.5 were determined in cutaneous nerve fibers of controls and subjects with DN. NOX5 methylation status and protein levels were assessed in subjects with DN that were divided into two groups based on changes in sural nerve myelinated fiber density: regenerators (showing significant nerve regeneration) and degenerators (showing significant nerve degeneration). NOX5 expression was present in deep myelinated cutaneous fibers. Bisulfite sequencing revealed that the NOX5 promoter, enriched with CpG sites, is hypomethylated in sural nerve biopsies of the degenerator cohort compared to the regenerator. The increase in NOX5 expression in degenerator sural nerves was accompanied with a decrease in MBP levels relative to the regenerator group. Our results highlight that in subjects with DN with sural nerve degeneration, there is hypomethylation of the NOX5 promoter associated with an increase in its protein expression, suggesting a potential epigenetic and mechanistic role of NOX5 in DN.

Disclosure
S. Eid: None. J.M. Hayes: None. K. Guo: None. C. Pacut: None. F. Mendelson: None. J. Hur: None. E.L. Feldman: None.
IMG-1 Activates and Mobilizes Insulin-Producing Islet Progenitor Cells (CD133+/Insulin+) In Vitro
Authors: BOTTINO RITA , POLLETT JONATHAN B. , THAI NGOC , TRUCCO MASSIMO 
Published: June 2018

IMG-1 is a novel therapeutic for the treatment of types 1 and 2 diabetes that may have islet regenerative properties. 300 islets isolated from mouse pancreases were cultured in standard media with IMG-1 (0.5µg/mL) and compared with control non-treatment. Within 24 hours, the IMG-1 treated islets became smaller (less clustered) and islet cells appeared to actively migrate away from the clusters. Control islets maintained their clustered morphology and did not show any evidence of cell migration. Three days after IMG-1 treatment, there were both free-floating and attached cells in the culture. The free-floating cells were 30% viable and the attached cells and leftover islets were 85% viable. In contrast, control islet cultures had neither free-floating nor attached cells although islet clusters looked healthy (cell viability &gt;90%). By day 10 of IMG-1 treatment, islet clusters were few in numbers (&lt;10%), no free-floating cells remained, and the attached cells appeared to form colonies. Total cell viability was &gt;90%. Control cells still had no migration or attachment, although within the islet clusters cell viability remained around &gt;90%. At 25 days, islet cells from both cultures were fixed and stained for the progenitor cell marker, CD133, and insulin. IMG-1 treated colonies were 75% insulin-positive by immunohistostaining. Furthermore, these cells were 70% CD133-positive with 60% of the culture being CD133/Insulin double-positive. In contrast, non-treated islets were 90% insulin+ cells, but &lt;5% CD133+ and &lt;5% double positive (CD133/insulin). IMG-1 has a novel effect on islets and induces the activation and migration of islet cells out of the islet. These cells form colonies of CD133/insulin double-positive cells that putatively represent activated beta-cell progenitor cells that can be used to regenerate damaged islets.

Disclosure
R. Bottino: None. J.B. Pollett: Employee; Self; Imagine Pharma. N. Thai: Board Member; Self; Imagine Pharma. M. Trucco: None.
Thymic Development of Beta-Cell Specific Regulatory T Cells in Neonatal NOD Mice
Authors: LIN YONG , LEE THOMAS , BETTINI MARIA , BETTINI MATTHEW 
Published: June 2018

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that involves the destruction of insulin producing β cells found within the pancreatic islets of Langerhans. Recent attention to neonatal thymocyte development has revealed a window during which central tolerance is established in an Autoimmune Regulator (Aire) dependent manner. However, expression of Chromogranin A (ChgA) within the thymus has not been detected; therefore, tolerance to ChgA may be due in part to peripheral dendritic cells (DCs) presenting ChgA to developing thymocytes. The relative contribution of thymically and peripherally derived antigens in the generation of neonatal β cell antigen specific CD4+ regulatory T cells (Tregs) is not well understood. For example, post-translational modification (PTM) of ChgA by fusion with an insulin peptide increases epitope immunogenicity by forming neo-antigens. Pathogenic T cells that are specific for neo-antigens uniquely expressed in the pancreas may escape thymic selection. Therefore, we utilized two experimental designs to test the role of peripheral antigen exposure on neonatal development of β cell specific Tregs. Firstly, we adoptively transferred dendritic cells pulsed with ChgA peptides into BDC2.5 TCR Tg mice and WT NOD mice to study the role of migratory DCs on neonatal development of ChgA Tregs. Secondly, we targeted thymic Langerin+ dendritic cells with anti-Langerin linked to ChgA peptides. In both experimental approaches, we found a robust increase in the ratio and number of ChgA specific Tregs in the presence of the hybrid insulin/chromogranin A peptide (2.5HIP) while also observing an increase in negative selection. The expansion of thymic Tregs was observed in both adult and neonatal mice. Together our data suggests neonatal exposure to PTM peptides enhances thymic development of β cell specific Tregs and may alter T1D pathogenesis.

Disclosure
Y. Lin: None. T. Lee: None. M. Bettini: None. M. Bettini: None.
Toll-Like Receptors 2/4 and Diabetic Peripheral Neuropathy
Authors: ELZINGA SARAH , MURDOCK BENJAMIN , HAYES JOHN M. , TABBEY MAEGAN A. , FELDMAN EVA L. 
Published: June 2018

Peripheral neuropathy (PN) is a common, morbid complication of diabetes and prediabetes. Dysfunction of toll like receptors (TLR) 2 and 4 has been implicated in PN pathogenesis, and blocking TLR4 is proposed as a treatment for neuropathic pain.
WT and TLR 2/4 global KO (TLR2/4-/-; n=5) mice were placed on a 60% high fat diet (HFD, n=5), with 5 WT controls provided a standard diet (CON, n=5). Mice underwent metabolic, neuropathic, and immunological phenotyping (Table 1) at early and late disease time points.
TLR2/4-/- mice weighed more (P ≤ 0.023) but did not have increased fasting blood glucose levels. They had reduced hind paw withdrawal latencies (HPL) compared to HFD mice (P ≤ 0.01) and were similar to CON in early disease, but worsened in later disease. TLR2/4-/- mice consistently had lower sural nerve conduction velocities than CON and worsened as disease progressed (P ≤ 0.048). Immunophenotyping during late disease showed differences in the number of peripheral blood Ly6C- myeloid cells (P ≤ 0.05) and F4/80+ expression (P ≤ 0.0001). Inflammatory profiles of the sural nerve indicated a lower gene expression of the chemoattractant CCL-12 chemokine in TLR2/4-/- compared to CON mice.
These results suggest that TLR2/4 KO may be initially protective against HFD induced neuropathy, but as PN progresses this effect is lost. Differences in circulating immune cells and CCL-12 gene expression may indicate an anti-inflammatory phenotype in TLR2/4-/- mice.Table 1. Metabolic, neuropathic and immunological phenotyping.Phenotypic MetricCON (n=5)HFD (n=5)TLR2/4-/-(n=5)Late stage weight (g)26 ± 339 ± 351 ± 3Late stage fasting blood glucose (mg/dL)150 ± 22190 ± 22172 ± 22HPL (sec)Early: 3.6 ± 0.3Late: 3.5 ± 0.4Early: 5.7 ± 0.3Late: 5.5 ± 0.4Early: 4.4 ± 0.3Late: 6.1 ± 0.4Sural NCV (m/s)Early: 22.0 ± 0.7Late: 23.9 ± 0.7Early: 18.5 ± 0.7Late: 17.9 ± 0.7Early: 20.0 ± 0.7Late: 17.2 ± 0.9Late stage Ly6C- circulating myeloid cells (104 cells/mL)7.8 ± 2.614.0 ± 2.74.8 ± 2.7Late stage circulating myeloid F4/80 expression (mean florescent intensity)978 ± 9611383 ± 10199967 ± 1019Late stage CCL-12 sural nerve gene expression (relative quantity)1.16 ± 0.240.59 ± 0.280.32 ± 0.24

Disclosure
S. Elzinga: None. B. Murdock: None. J.M. Hayes: None. M.A. Tabbey: None. E.L. Feldman: None.
CD70 Modulates Thymocyte Development and Type 1 Diabetes in NOD Mice
Authors: YE CHENG , DRIVER JOHN , WILES MICHAEL V. , SERREZE DAVE 
Published: June 2018

CD70 is a tumor necrosis factor (TNF) superfamily member expressed by activated T cells, B cells, dendritic cells, NK cells and mature macrophages. It interacts with CD27, a TNF receptor family member on T cells that regulates T cell activation, proliferation and survival. Type 1 diabetes (T1D) is caused by T cell mediated beta cell destruction, however, T1D-prone NOD mice developed accelerated disease in the absence of CD70. This was associated with altered proportions of double negative vs. double positive thymocytes in CD70 knockout mice, indicating that CD70 modulates global thymopoiesis. Also affected, but in opposing directions, was the development of regulatory T cells (Treg) and natural killer T (NKT) cells, two immunoregulatory lymphocyte subsets that are functionally and numerically deficient in NOD mice. Tregs frequencies were reduced by 23% and 32% in CD70 heterozygous (CD70-/+) and homozygous (CD70-/-) knockout mice. In contrast, NKT cell frequencies were respectively increased by 48% and 61% in CD70-/+ and CD70-/- mice compared to standard NOD mice. The higher level of NKT cells in CD70 knockout mice was unexpectedly associated with reduced levels of thymic CD1d, a molecule that is required for NKT cell development. Collectively, our results show that CD70 controls the development of several T cell populations that are known to modulate T1D development in NOD mice and that, in the absence of CD70, T1D is exacerbated. These finding may facilitate the development of clinical therapeutics aimed at enhancing the stimulatory function of CD70 as a way to suppress disease.

Disclosure
C. Ye: None. J. Driver: None. M.V. Wiles: None. D. Serreze: None.
Adiponectin Increases Glucose-Dependent Electrical Activity and Insulin Secretion in Mouse Pancreatic ß Cells
Authors: REAL JOANA I. , GARCIA BELEN CHANCLON , ASTERHOLM INGRID WERNSTEDT , RORSMAN PATRIK 
Published: June 2018

Decreased plasma adiponectin has been implicated as a cause of diabetes. However, the functional impact of adiponectin on glucose-dependent responses of β-cells remains poorly understood. Here we have investigated whether chronically and acutely increased adiponectin levels affect electrical activity (sharp electrodes) and insulin secretion in β-cells using respectively, adiponectin overexpressing (APNtg) mice and acute application of adiponectin. In the acute exposure experiments, adiponectin (20 µg/ml, 11 mM glucose) increased insulin secretion 42% and β-cell electrical activity (measured as the fraction ‘active phase’) 32%. In islets from APNtg mice insulin secretion increased 44% and active phase increased 25%. The effects on electrical activity in APNtg β-cells correlated with membrane hyperpolarization and the resting membrane potential at 0 mM glucose averaged -60±2 in control and -69±2 in APNtg β-cells. There was a moderate effect on action potential width (&lt;20%) and a more pronounced impact on action potential frequency by exogenous adiponectin (+35%) and in APNtg β-cells (+50%).These results suggest that both acutely and chronically increased adiponectin levels have a positive effect on electrical activity and insulin secretion of pancreatic β-cells.

Disclosure
J.I. Real: None. B. Chanclon Garcia: None. I. Wernstedt Asterholm: None. P. Rorsman: None.
Recombinant Probiotics Expressing Angiotensin-(1-7) Improves Glucose Metabolism and Diabetes-Induced Renal and Retinal Injury
Authors: LI QIUHONG , XU KANG , DU TAO , ZHU PING , VERMA AMRISHA 
Published: June 2018

Purpose: Multitude of animal studies substantiate the beneficial effects of Ang-(1-7) in diabetes and associated complications. However, the biggest impediment to translate this into clinical application is large-scale production of high quality Ang-(1-7) with enhanced tissue bioavailability. As emerging evidence also implicates the beneficial effects of probiotics in managing diabetes, this study aimed to test the hypothesis that oral delivery of recombinant probiotics expressing Ang-(1-7) will improve glucose metabolism and diabetes-induced tissue damage in animal models.
Method: Ang-(1-7) was expressed as a secreted fusion protein with a transepithelial carrier in Lactobacillus paracasei (LP) to allow uptake into circulation and target tissues. Adult diabetic eNOS-/- mice were orally gavaged daily with 1x109 CFU of LP secreting Ang-(1-7) (LP-A), or LP alone for eight weeks. Glucose metabolism and tissues were assessed using standard assays and histochemical techniques.
Result: Oral feeding of LP-A significantly lowered fasted-state blood glucose (P=0.042); improved the intraperitoneal glucose tolerance test (P=0.034) than control mice. LP-A-treated mice showed higher plasma insulin level (P=0.028) and insulin expression in islets β cells compared to control mice. LP-A treatment significantly reduced apoptotic cell death in kidney, improved diabetes-induced collagen deposits in the glomerular tuft and the tubular epithelia in diabetic mice. LP-A treatment also significantly reduced retinal gliosis, inflammation, neuronal cell death and loss of retinal vascular capillaries.
Conclusion: Oral administration of recombinant probiotics secreting Ang-(1-7) improved glucose tolerance by enhancing insulin level, and reduced diabetes-induced damage in kidney and retina. Thus, recombinant probiotics-based delivery of Ang-(1-7) may hold promising therapeutic potential for diabetes and associated complications.

Disclosure
Q. Li: None. K. Xu: None. T. Du: None. P. Zhu: None. A. Verma: None.