bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrative Analysis of Human Plasma Proteome/Metabolome Reveals Central Role for Phospholipid, Retinol, and Histidine Metabolism after Gastric Bypass
Authors: DREYFUSS JONATHAN , YUCHI YIXING , PAN HUI , SIMONSON DONALD C. , VERNON ASHLEY H. , ARYAL PRATIK , KAHN BARBARA , FOSTER KATHLEEN , KASIF SIMON , GOLDFINE ALLISON , PATTI MARY E.
Published: June 2018

Bariatric surgery has emerged as a potent approach to improve obesity-related metabolic comorbidities including type 2 diabetes (T2D). However, molecular mechanisms responsible for metabolic improvement after bariatric surgery remain incompletely understood. We profiled and integrated the fasting plasma proteome (aptamer-based Somalogic platform) and metabolome (MS, Metabolon) from patients with T2D at baseline and up to 3 years after randomization to either gastric bypass (GB) or nonsurgical diabetes/weight management (DMW) within the SLIMM-T2D longitudinal clinical trial (random subset, n=19 per group). To identify top-ranking pathways encompassing both proteomic and metabolomic data, we developed a systems approach employing two interdependent programs: ezlimma (for differential analysis) and Pathway Analysis via Network Smoothing (PANTS). PANTS smooths differential statistics over a large network of interacting proteins/metabolites, calculating significance by permutation. Top-ranking pathways were phospholipid (e.g., choline), retinol (e.g., retinol-binding protein 4, RBP4), and histidine metabolism (e.g., carnosine dipeptidase 1, CNDP1). Quantitative Western blot confirmed 26% reduction in RBP4 in GB vs. DWM; ELISA confirmed 66% reduction in CNDP1 in GB vs. DWM (both p<0.05, 3 months). CNDP1 change at 3 months was associated with improved metabolism at 1 year (BMI r=0.67, p<10-5; HbA1c r=0.61, p<10-4). Causal inference analysis in the entire cohort identified CNDP1 change at 3 months as the most likely protein to cause improved HbA1c at 1 year (p=0.01). Decreased CNDP1 occurred before significant weight loss, suggesting weight-independent impact on glycemia. Investigating modulation of CNDP1 activity, its enzymatic products Ala/His, and related metabolites will be important to determine if these pathways may serve as targets for T2D therapy.

Disclosure

J. Dreyfuss: None. Y. Yuchi: None. H. Pan: None. D.C. Simonson: Advisory Panel; Self; GI Windows, Inc.. Stock/Shareholder; Self; GI Windows, Inc.. Stock/Shareholder; Spouse/Partner; Phase V Technologies, Inc. A.H. Vernon: None. P. Aryal: None. B. Kahn: Advisory Panel; Self; Janssen Research & Development. Research Support; Self; Janssen Research & Development. Advisory Panel; Self; Alterna Biotech. K. Foster: None. S. Kasif: None. A. Goldfine: Employee; Self; Novartis AG. M.E. Patti: Research Support; Self; Janssen Pharmaceuticals, Inc.. Other Relationship; Self; Xeris Pharmaceuticals, Inc.. Research Support; Self; Ethicon US, LLC., Coviden, MedImmune. Other Relationship; Self; Novo Nordisk Inc., XOMA Corporation, AstraZeneca, Nestlé. Research Support; Self; Dexcom, Inc.. Consultant; Self; Eiger BioPharmaceuticals.
HLA-C Position 275, a Novel HLA Locus Identified in Autoantibody-Positive Type 1 Diabetes of Chinese Han Population Based on a Genome-Wide Association Study
Authors: XU KUANFENG , CHEN YANG , GU YONG , ZHU MENG , ZHANG MEI , SUN MIN , XU XINYU , HSU HSIANG-TING , CHEN HENG , SHI YUN , CAI YUN , DAI HAO , ZHENG SHUAI , ZHENG XUQIN , ZHOU HONGWEN , YU LIPING , HU ZHIBIN , ZHOU ZHIGUANG , WENG JIANPING , SHEN HONGBING , YANG TAO
Published: June 2018

Type 1 diabetes (T1D) is a highly heritable autoimmune disease. Although more than 50 non-HLA risk loci have been identified, the strongest associations were observed in HLA regions in Caucasian populations. The aim of this study is to identify T1D risk HLA loci in Chinese Han population and explore the risk prediction models based on these loci. A two-stage genome-wide association study (GWAS) of T1D (at least one autoantibody positive) was performed in Chinese Han population, the GWAS scan was conducted by the Illumina Human OmniZhongHua platform and included 1,045 T1D cases and 1,3controls, and the replication included 1,378 cases and 3,774 controls. Promising associations were further validated by combining with the data from the Wellcome Trust Case Control Consortium (WTCCC). Association analyses for T1D used logistic regression models assuming additive effects. Results from different stages were combined with fixed effects model in meta-analysis. In the GWAS scan stage, we identified 18 distinct genomic regions with P-values <1.00×10-5. As expected, the strongest associations with T1D were located in the HLA region, and rs1770 at MHC (OR=4.54, P<1.00×10-324), a previously identified Caucasian T1D risk locus, showed the strongest association after combining the results from different stages. Further fine mapping in the HLA region identified five independent risk loci, a novel Chinese specific locus HLA-C position 275 (P=9.78×10−12) was discovered except for rs1770, HLA-DRB1 position 11, HLA-DRB1 position74, and HLA-A position 9. Risk prediction model based on these five independent loci of HLA can achieve an AUC of 0.85 (0.84-0.87). A s the primary T1D GWA study in Asian population to date, the results identified a Chinese specific HLA locus and would be the basis of precision medicine of Chinese T1D by risk prediction based on the identified HLA loci.

Disclosure

K. Xu: None. Y. Chen: None. Y. Gu: None. M. Zhu: None. M. Zhang: None. M. Sun: None. X. Xu: None. H. Hsu: None. H. Chen: None. Y. Shi: None. Y. Cai: None. H. Dai: None. S. Zheng: None. X. Zheng: None. H. Zhou: None. L. Yu: None. Z. Hu: None. Z. Zhou: None. J. Weng: None. H. Shen: None. T. Yang: None.
Unraveling the Athlete’s Paradox—Higher Insulin Sensitivity and Lower PKC? Activation Despite Higher Bioactive Lipids in Endurance-Trained Athletes
Authors: PESTA DOMINIK , ANADOL-SCHMITZ EVRIM , GANCHEVA SOFIYA , MARKGRAF DANIEL F. , ZAHARIA OANA P. , KATSUYAMA HISAYUKI , KUPRIYANOVA YULIYA , HWANG JONG-HEE , ZHANG DONGYAN , SHULMAN GERALD I. , RODEN MICHAEL
Published: June 2018

High intramyocellular lipid (IMCL) content associates negatively with insulin sensitivity (IS) in insulin resistant, but not in endurance-trained humans. It has been hypothesized that different cellular distribution of bioactive lipids such as diacylglycerols (DAG) and ceramides (CER) could interfere with insulin action and underlie this “athlete’s paradox.” We examined endurance-trained athletes (ATH; n=9) and sedentary individuals (SED; n=12) with comparable total IMCL, as measured by 1H-magnetic resonance spectroscopy, who underwent spiroergometry and hyperinsulinemic-euglycemic clamps to assess maximal oxidative capacity and IS, respectively. In skeletal muscle biopsies, translocation of protein kinase C (PKC) θ and ε were determined by Western blotting and concentrations of DAG and CER were measured using targeted LC-tandem mass spectrometry upon separating fractions of cellular membranes, lipid droplets and cytosol by ultracentrifugation. Maximal oxidative capacity and IS were 46% and 47% higher in ATH than in SED (both p<0.01), respectively. The membrane:cytosol ratio of PKCθ, which reflects PKCθ activity, was 62% lower in ATH consistent with their increased IS (p<0.01), while there was no group difference for PKCε. Total and membrane DAG (40% and 48%, both p<0.01) as well as membrane CER (15%, p<0.05) were higher in ATH while the respective concentrations in lipid droplets and cytosol did not differ. In SED, IS correlated inversely with all stereoselective subspecies of lipid-droplet DAG. On the other hand, cytosolic sn-1,2 (C16:0-C18:2) and sn-1,3 (C18:1-C18:0) DAG correlated positively with IS in ATH.

In conclusion, higher IS in endurance-trained ATH can be explained by lower muscle PKCθ activation, which may be due to differences in the stereoselectivity and/or subcompartmentation of cellular DAG between ATH and insulin resistant SED.

Disclosure

D. Pesta: None. E. Anadol-Schmitz: None. S. Gancheva: None. D.F. Markgraf: None. O.P. Zaharia: None. H. Katsuyama: None. Y. Kupriyanova: None. J. Hwang: None. D. Zhang: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc., Novo Nordisk Inc.. Research Support; Self; Gilead Sciences, Inc. M. Roden: Speaker's Bureau; Self; Boehringer Ingelheim GmbH. Research Support; Self; Boehringer Ingelheim GmbH. Consultant; Self; Poxel SA. Research Support; Self; Danone Nutricia Early Life Nutrition, GlaxoSmithKline plc., Nutricia Advanced Medical Nutrition, Sanofi.
HuR Influences Metabolic Flexibility in Skeletal Muscle
Authors: WARFEL JAYCOB D.
Published: June 2018

Metabolic Flexibility allows for the oxidation of fuels based on their availability. Respiratory Quotient (RQ) values from subjects with type 2 diabetes mellitus in both basal and insulin stimulated states remain consistent, indicating a decreased ability to switch between available substrates. The molecular mechanisms governing defects in this switching ability are unclear at present. We therefore compared global gene expression in skeletal muscle between obese subjects classified by their sleep and 24 hour RQ as either metabolically flexible or inflexible. From this data we identified a number of transcripts stabilized by the RNA binding protein, HuR, which were enriched in metabolically flexible subjects. This finding prompted us to generate mice with a skeletal muscle specific knockout of Elavl1, the gene encoding HuR (HuRm-/-). HuRm-/- mice have a metabolically inflexible phenotype with mild obesity and impaired glucose tolerance compared to control littermates. HuRm-/- mice have an increase in RER; and decreased expression of genes involved in oxidative phosphorylation, fatty acid transport, and mitochondrial fatty acid metabolism, suggesting a decreased ability to use fats as an energy source. HuRm-/- mouse skeletal muscle also shows decreases in protein levels of important modulators of the mTOR signaling pathway, and compensatory increases in transcript levels of HuR target mRNAs. These data highlight the importance of HuR function in skeletal muscle metabolism; and suggest HuR as a mediator of metabolite selection.

Disclosure

J.D. Warfel: None.
Reprograming of Human Adipocytes to a Briter Phenotype—Enhanced Fatty Acid Oxidation and Lipid Droplet Remodeling
Authors: LEE MI-JEONG , JASH SUKANTA , PURI VISHWAJEET , FRIED SUSAN K.
Published: June 2018

Converting energy storing white adipocytes to energy wasting ‘brite’ ones is now recognized to be feasible and may confer protection against obesity and its related cardiometabolic diseases. Fragments of omental and subcutaneous human adipose tissues were cultured for 7 days with insulin and dexamethasone with or without rosiglitazone (rosi) to induce britening. Rosi caused a metabolic reprograming of the adipocytes including increased rates of basal FA oxidation and the expression of mRNAs and proteins in multiple pathways that modulate FA metabolism, triacylglycerol synthesis, cellular FA trafficking, and mitochondrial oxidative capacity (PGC1a, UCP1, CIDEA, PLIN5, FABP3). Furthermore, these changes were associated with a dramatic remodeling of the surface of the adipocyte lipid droplet (LD) such that clusters of small LDs decorated with mitochondria were formed. Formation of these small LDs was blocked by inhibition of lipolysis and required FA activation and reesterification. Surprisingly, both omental and subcutaneous adipocytes were similarly ‘britened’. A similar remodeling was induced in primary cultures of newly-differentiated human adipocytes and these cells showed higher rates of basal and maximally-stimulated oxygen consumption. Consistent with transcriptome changes in both systems, lipidomics showed increased levels of unsaturated FAs, phospholipid species, and cardiolipins in brite cells. Taken together, these results identify a coordinated metabolic and structural reprogramming of mature human adipocytes toward a more oxidative phenotype. Although rosi has unacceptable clinical side effects, knowledge of the downstream effectors of this metabolic improvement may lead to development of targeted approaches to improve adipocyte and metabolic health and prevent type 2 diabetes in human obesity.

Disclosure

M. Lee: None. S. Jash: None. V. Puri: None. S.K. Fried: None.
Effects of Acute Experimental Hyperglycemia on Oxidative Markers and AGE-RAGE Dynamics in Obese Humans
Authors: PERKINS RYAN , MIRANDA EDWIN R. , KARSTOFT JR. KRISTIAN , BEISSWENGER PAUL J. , SOLOMON THOMAS P. , HAUS JACOB M.
Published: June 2018

Chronic hyperglycemia promotes oxidative stress and advanced glycation end product (AGE) formation, leading to recognition by the AGE receptor (RAGE). AGE-RAGE binding and inflammation perpetuates diabetic complications, however the study of these phenomenon present inherent challenges in vivo. Therefore, our purpose was to evaluate the effects of acute experimental hyperglycemia on AGEs, oxidative markers, and RAGE expression in healthy, obese subjects (n=10; 31.2±1.2 kg/m2; 56±3 y) subjected to hyperglycemic clamps (+5.4 mM above basal). Plasma was assayed at baseline, 2, and 24 h post glucose-infusion for well-known AGE-free adducts (CML, CEL, G-H1, MG-H1, 3DG-H) and oxidative markers (MethSO, AAA) via LC-MS/MS as well as soluble RAGE (sRAGE) isoforms via ELISA. Urine was also assayed (basal and 24 h) for AGEs and oxidative markers (normalized to creatinine) and skeletal muscle biopsies were used to study RAGE expression via Western blot. Most circulating factors (MethSO, AAA, CEL, MG-H1, and G-H1) decreased (p<0.05) over time, while CML increased (p<0.05) and 3DG-H remained unchanged (p>0.05). In urine, only MethSO was seen to increase at 24 h (p<0.05). Plasma:Urine ratios were decreased (p<0.05) for MethSO, AAA, CML, MG-H1, while ratios were increased (p<0.05) for CML. All species of sRAGE isoforms (total, cleaved and esRAGE) were unchanged (p>0.05). In skeletal muscle tissue, there was a trend (p<0.1) for decreased RAGE protein expression at 2 h which returned to baseline at 24 h. This study demonstrates AGEs and oxidative markers are sensitive to the physiological dynamics of acute hyperglycemia in obese, but otherwise healthy humans. The differential findings presented here were surprising and may be explained by increased AGE-RAGE binding or AGE sequestration, osmotic diuresis and/or oxidative defense mechanisms. These findings are relevant for postprandial glucose excursions and highlights the importance of appropriate glucose management.

Disclosure

R. Perkins: None. E.R. Miranda: None. K. Karstoft: None. P.J. Beisswenger: Employee; Self; PreventAGE Health Care, LLC.. T.P. Solomon: None. J.M. Haus: None.
Targeting the Amyloid Precursor Protein (APP) to Mitochondria of White Adipose Tissues Triggers Mitochondrial Dysfunction and Obesity
Authors: AN YU A. , SCHERER PHILIPP E.
Published: June 2018

The role of mitochondrial dysfunction in white adipose tissues (WAT) under different metabolic conditions is not well understood. Here, we report that under high fat diet (HFD) conditions, endogenous production of APP is significantly induced and, instead of being secreted, the protein is highly enriched in WAT mitochondria. To better understand the role of APP, we established an inducible model by directing the full-length APP with a mitochondrial targeting presequence quantitatively to mitochondria, selectively in adipocytes. We validated that APP mRNA and protein levels were significantly elevated in subcutaneous WAT, and the protein was markedly accumulated in isolated mitochondria from sWAT. EM imaging suggested widespread loss of mitochondrial cristae. Respiratory measurements on a Seahorse instrument revealed a rapid impairment in mitochondrial functions in sWAT, concomitant with an increase in adipocyte size within a few days. APP transgenic mice displayed significantly enhanced fat mass, along with impaired glucose tolerance and reduced insulin sensitivity under a long term HFD challenge. By H and E staining, we observed widespread inflammatory cell infiltration in WAT, and consistent with the adipose tissue dysfunction, we also noticed massive hepatic steatosis. Adiponectin levels in plasma and sWAT were significantly reduced. We specifically eliminated endogenous APP in WAT through a conditioned knock-out mouse model (Appflox/flox) to demonstrate that adipocyte-specific deletion of APP can ameliorate high fat diet-induced mitochondrial dysfunction.

In summary, accumulated APP in WAT mitochondria significantly induces mitochondrial dysfunction in adipocytes, leading to unhealthy adipose tissue expansion and eventually resulting in obesity and systemic insulin resistance.

Disclosure

Y.A. An: None. P.E. Scherer: None.
HOXC10 Maintains White Adipose Tissue Identity and Its Loss Leads to Enhanced Browning of White Adipose Tissues
Authors: TAN ANGELINE H.Y. , GUN SINYEE , HAN WEIPING
Published: June 2018

Increased thermogenesis via increased beige fat development enhances energy expenditure and improves glucose homeostasis. However, thermogenic mechanisms regulating beige adipogenesis within white adipose tissue (WAT) are less understood. The homeobox-containing transcription factor, HOXC10, is traditionally thought to regulate musculoskeletal and patterning of limb development in mammals. We found that HOXC10 is expressed in subcutaneous WAT. Ectopic expression of HOXC10 in adipocytes suppressed brown markers genes, while knockdown of HOXC10 upregulates brown genes expression. To investigate the functional role of HOXC10 in adipose tissue, we generated adipose-specific HOXC10 knockout (AHKO) mice using adiponectin-Cre mice. We show that development of functional beige adipocytes within subcutaneous WAT is enhanced in AHKO mice, contributing to decreased white fat in the lean AHKO mice kept at room temperature. Furthermore, AHKO mice show reduced weight gain, lower fat mass and improved glucose tolerance compared to wild type (WT) mice when fed on high fat diet. Interestingly, AHKO mice exhibit enhanced thermogenic response to cold exposure. These mice also show increased energy expenditure, alongside increased in brown fat markers in subcutaneous WAT, with no effect on classical BAT. These data indicate that HOXC10 plays a critical role in maintaining white adipose tissue identity, and loss of HOXC10 promotes browning in WAT.

Disclosure

A.H.Y. Tan: None. S. Gun: None. W. Han: None.
Growth Differentiation Factor-15 (GDF-15) Inhibits Gastric Emptying in Rodents as Part of Its Anorectic Mechanism of Action
Authors: HINKE SIMON A. , CAVANAUGH CASSANDRE R. , KIRCHNER THOMAS , LANG WENSHENG , MENG RONG , WALLACE NATHANIEL H. , LIU JR. JIANYING , D'AQUINO KATHARINE E. , HO GEORGE , RANKIN MATTHEW M. , WANG SHENG-PING , CHAVEZ JOSE A. , NELSON SERENA M. , FURMAN JENNIFER , MULLICAN SHANNON , RANGWALA SHAMINA M. , NAWROCKI ANDREA R.
Published: June 2018

GDF-15 is a secreted circulating polypeptide that regulates systemic energy balance. GDF-15 agonists may have therapeutic potential as anorectic agents in obesity and type 2 diabetes. The receptor for GDF-15, Gfral, is expressed on specific neurons in the area postrema (AP) of the hindbrain, and is necessary for the effect of GDF-15 on food intake. Given the role of the AP in vagal control of gastric motility, we sought to investigate the potential effects of GDF-15 on gastric emptying. Food intake reduction by GDF-15 was confirmed in C57Bl/6N mice using BioDAQ continuous food consumption monitoring. Animals were treated sc with recombinant His-tagged human GDF-15 prior to initiation of the dark cycle; GDF-15 treated mice showed significant reduction in food intake relative to vehicle treated controls (12 hour cumulative food intake: 3nmol/kg, -19.9±10.5%; 10nmol/kg, -58.0±10.0%; P<0.0001, n=8). Gastric emptying was assessed using an oral acetaminophen (AAP) absorption test over 90 minute using LC/MS detection. This method was validated using known inhibitor of gastric emptying, Exendin-4 as a positive control; 7.2nmol/kg Exendin-4 significantly reduced integrated AAP absorption by -44.7±5.1% (P<0.01, n=8). In this assay, GDF-15 caused a significant dose-dependent inhibition of gastric emptying, reducing acetaminophen AUC levels by -18.1±10.6% at 1nmol/kg, and by -36.0±9.9% at 10nmol/kg, relative to vehicle treated control mice (P<0.01, n=8). Comparable results were obtained in an independent repetition of the study. We extended the results obtained in mice to SD rats, where we similarly observed a significant reduction in gastric emptying following GDF-15 treatment. Hence, GDF-15 appears to reduce gastric emptying rate in both mouse and rat, potentially contributing to the food intake suppression mechanism of action.

Disclosure

S.A. Hinke: Employee; Self; Janssen Research & Development. C.R. Cavanaugh: Employee; Self; Janssen Research & Development. T. Kirchner: None. W. Lang: None. R. Meng: None. N.H. Wallace: None. J. Liu: None. K.E. D'Aquino: Employee; Self; Janssen Research & Development. Employee; Spouse/Partner; Janssen Research & Development. G. Ho: None. M.M. Rankin: Employee; Self; Janssen Research & Development. S. Wang: None. J.A. Chavez: None. S.M. Nelson: None. J. Furman: Employee; Self; Janssen Research & Development. S. Mullican: Employee; Self; Janssen Research & Development. S.M. Rangwala: Employee; Self; Janssen Research & Development. A.R. Nawrocki: Employee; Self; Janssen Pharmaceuticals, Inc..
Sex-Specific Role of Adipose-Derived Lipocalin-2 in Diet-Induced Obesity and Insulin Resistance
Authors: KRISHNAN KARTHICKEYAN CHELLA , SABIR SIMON , FLOYD RAQUEL , JAYASEKERA DULSHAN , LUSIS ALDONS
Published: June 2018

Lipocalin-2 (LCN2) is a 25-kDa secretory adipokine that is up regulated in adipose tissues of genetically obese animals and humans. LCN2 was also proposed to regulate insulin resistance (IR) in animal studies. Besides, we have shown adipose Lcn2 expression to be negatively regulated by estradiol using a mouse reference population of over ∼100 strains, the Hybrid Mouse Diversity Panel (HMDP). Recently, LCN2 has also been implicated in recruiting neutrophils to the liver in both alcoholic and nonalcoholic steatohepatitis. To further explore the causal role of LCN2 in diet-induced obesity and its metabolic complications, we carried out systems genetics analyses on multi-omics data collected from both males and females of HMDP fed a high fat/high sucrose (HF/HS) diet: Genotypes, 200,000 high-resolution SNPs; Phenotypes, obesity (fat mass, and weight of three visceral adipose depots), IR (fasting levels of glucose, insulin and HOMA-IR) and steatosis (liver triglyceride levels); and Transcriptomics, mRNA microarrays of adipose and liver tissues. Our analyses revealed female, but not male, adipose-derived LCN2 is mechanistically linked to deregulated adiposity and enhanced insulin resistance. To functionally validate the causal role of female adipose LCN2, we conditionally overexpressed either LCN2 or GFP in adipose tissues of mice of both sexes by adeno-associated viral vectors and subjected them to HF/HS diet. We demonstrated deregulated metabolic homeostasis: increased adiposity (fat mass), glucose intolerance (GTT) and insulin resistance (ITT and HOMA-IR) only in females. Follow-up ex vivo bioenergetics studies in both adipose and liver tissues demonstrated deregulated mitochondrial respiration (lowered in adipose; enhanced in liver) mediated by LCN2 overexpression only in females, thereby revealing mitochondrial dysfunction as one of the key mechanism(s) mediated by LCN2 in diet-induced obesity and its complications.

Disclosure

K. Chella Krishnan: None. S. Sabir: None. R. Floyd: None. D. Jayasekera: None. A. Lusis: None.
IKKß and Wnt/ß-Catenin Signaling Interaction Regulates Adipogenesis and Osteogenesis in Mesenchymal Stem Cells of Nonhuman Primates and Humans
Authors: SUI YIPENG , PARK SE-HYUNG , KERN PHILIP A. , TEMEL RYAN , ZHOU CHANGCHENG
Published: June 2018

IκB kinase β (IKKβ), a central coordinator of inflammation through activation of NF-κB, has been implicated in the pathogenesis of obesity-associated metabolic disorders. We recently demonstrated that IKKβ also functions in adipose progenitors to regulate adipogenesis and adipose tissue development in mice. Deficiency of IKKβ in adipose progenitors decreased high-fat (HF)-elicited adipogenesis and protected mice from diet-induced adiposity and insulin resistance. Here we report that IKKβ can regulate both adipogenesis and osteogenesis in mesenchymal stem cells (MSCs) of non-human primates (Macaca fascicularis) and humans. Activation or overexpression of IKKβ increased adipocyte differentiation but inhibited osteoblast differentiation of MSCs isolated from adipose tissue and bone marrow of macaques and humans. By contrast, IKKβ loss of function decreased adipogenesis and increased osteogenesis in macaque and human MSCs. Mechanistically, IKKβ can directly interact with Wnt/β-catenin signaling and phosphorylate β-catenin at serine-33, -37 and -45, leading to its ubiquitination and degradation. Inhibition of canonical Wnt/β-catenin signaling then contributed to the increased adipogenesis and decreased osteogenesis in MSCs. Our findings demonstrate IKKβ as a key molecule that regulates MSC differentiation, and provide a connection between IKKβ and Wnt/β-catenin signaling which may lead to new insights into the pathogenesis of obesity and osteoporosis.

Disclosure

Y. Sui: None. S. Park: None. P.A. Kern: None. R. Temel: Research Support; Self; Ionis Pharmaceuticals, Inc., Regulus Therapeutics Inc.. Stock/Shareholder; Self; Merck & Co., Inc., Ionis Pharmaceuticals, Inc., Resverlogix Corp.. C. Zhou: None.
Relationship between Amount and Type of Dietary Fat, Postprandial Glycemia, and Insulin Requirements in Type 1 Diabetes
Authors: BELL KIRSTINE , DUKE SALLY , ALEXANDER KYLIE M. , MCGILL MARGARET , WONG JENCIA , FULCHER GREGORY , TWIGG STEPHEN M. , BRAND-MILLER JENNIE , STEIL GARRY M.
Published: June 2018

ADA recommends individuals with type 1 diabetes (T1D) are taught to adjust insulin for dietary fat however optimal adjustments are not yet clear. This study aimed to determine 1) the relationship between the ‘amount’ and ‘type’ of dietary fat and glycemia and 2) the optimal insulin adjustments for dietary fat. Six adults with T1D using insulin pump therapy attended the research clinic on 9 to 12 occasions. On the first 6 visits, participants consumed meals containing 45g CHO with either 0g, 20g, 40g, or 60g fat and either saturated (SFA), monounsaturated (MUFA) or polyunsaturated (PUFA) fat. Insulin was dosed using individual insulin: carbohydrate ratio as a dual-wave 50/50% split over 2h. On subsequent visits, participants repeated the 20 to 60g fat meals with the insulin dose estimated using a model predictive bolus, with up to 2 repeats/meal until glycemic control achieved. With the same insulin dose, mean 5h incremental area under the curve (iAUC) was increased by 46%, 23% and 139% for the 3 fat loads relative to the 0g fat meal (0g: 509 ± 153; 20g: 742 ± 742; 40g: 626 ± 74; 60g: 1216 ± 434mmol/L.min; ns). The type of fat made small but non-significant differences to the 5h iAUC. To achieve glycemic control, on average, participants required 20-60% more insulin, delivered as a dual-wave over 1.25-2h. This study provides foundation for mealtime insulin dosing recommendations for dietary fat in T1D.

Disclosure

K. Bell: Other Relationship; Self; Novo Nordisk Inc.. S. Duke: None. K.M. Alexander: None. M. McGill: Advisory Panel; Self; Abbott. Speaker's Bureau; Self; AstraZeneca. Advisory Panel; Self; Merck Sharp & Dohme Corp.. Speaker's Bureau; Self; Merck Sharp & Dohme Corp. J. Wong: Speaker's Bureau; Self; Novo Nordisk A/S, AstraZeneca, Eli Lilly and Company. G. Fulcher: Other Relationship; Self; Novo Nordisk Inc., Janssen Scientific Affairs, LLC., Boehringer Ingelheim GmbH, Merck Sharp & Dohme Corp. S.M. Twigg: Advisory Panel; Self; Abbott. Consultant; Self; Abbott. Advisory Panel; Self; Novo Nordisk Inc., Sanofi-Aventis, AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company. Speaker's Bureau; Self; AstraZeneca, Merck Sharp & Dohme Corp.. Other Relationship; Self; Abbott. J. Brand-Miller: Board Member; Self; Glycemic Index Foundation. Other Relationship; Self; Hachette Australia. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. Other Relationship; Self; Pam Krauss Books. G.M. Steil: Consultant; Self; Profusa, Eli Lilly and Company.
A Plant-Based Diet Improves Beta-Cell Function and Insulin Resistance in Overweight Adult—A 16-Week Randomized Clinical Trial
Authors: KAHLEOVA HANA , TURA ANDREA , HILL MARTIN , HOLUBKOV RICHARD , BARNARD NEAL
Published: June 2018

Background: The aim of this study was to test the effect of a 16-week plant-based dietary intervention on beta-cell function and insulin resistance in overweight adults with no history of diabetes.

Methods: Participants (n=75) were randomized to follow a low-fat vegan diet (n=38) or to make no diet changes (n=37) for 16 weeks. At baseline and 16 weeks, beta-cell function was assessed during standard meal tests. Insulin secretory rate was calculated by C-peptide deconvolution, and beta-cell function was quantified with a mathematical model. HOMA-IR (The Homeostasis Model Assessment) index was used to assess insulin resistance while fasting. Repeated measure ANOVA was used for statistical analysis.

Results: A marked increase in glucose-stimulated insulin secretion was observed in the intervention group compared with controls (interaction between group and time, Gxt, p<0.001). HOMA-IR index fell significantly (p<0.001) in the intervention group (treatment effect -1.0 [95% CI, -1.2 to -0.8]; Gxt, p=0.004). Changes in HOMA-IR correlated positively with changes in body mass index (BMI) and visceral fat volume (r=0.34; p=0.009 and r=0.42; p=0.001, respectively). The latter remained significant after adjustment for changes in BMI (r=0.41; p=0.002). Changes in glucose-induced insulin secretion correlated negatively with BMI changes (r=-0.25; p=0.04), but not with changes in visceral fat.

Conclusions: A 16-week low-fat vegan dietary intervention improved beta-cell function and insulin resistance in overweight adults. The improvement in insulin resistance was related to loss of visceral fat, independent of changes in BMI.

Disclosure

H. Kahleova: None. A. Tura: None. M. Hill: None. R. Holubkov: Other Relationship; Self; Pfizer Inc., MedImmune. N. Barnard: None.
Improved Beta-Cell Differentiation and Function in Isolated Islets Established in Dynamic Culture to Reduce Hypoxia
Authors: AL-JAHDHAMI NAJWA A. , ANDERSON SCOTT J. , ALDIBBIAT ALI , SHAW JAMES A.
Published: June 2018

Background: Pancreatic islet transplantation effectively prevents severe hypoglycaemia in type 1 diabetes. Restoration of normal β-cell functional mass remains elusive and we have recently demonstrated loss of β-cell end-differentiation in clinical islet recipients. We hypothesised that reducing islet hypoxia in culture would restore β-cell differentiation and function.

Methods: MIN6 pseudo-islets (PI) and primary human islets (HI) were cultured in either static (SC) or horizontal rotation (RC) culture for 7 days. Hypoxia was detected by pimonidazole (Pim) adduct formation and hypoxia-induced gene expression (qPCR). β-cell end-differentiation was assessed by UCN3 expression and IF staining (urocortin-3+ insulin+ cells/total insulin+ cells) with function assayed by glucose-stimulated insulin secretion (GSIS).

Results: Hypoxia was confirmed in established PI (70±1.0% Pim+). This was reduced in rotation culture (Pim+: 15±1.5% RC vs. 80±2.5% SC; p<0.05) with reduced hypoxia-induced gene expression compared with adherent MIN6 (LDHA: 6.0±2.1 RC vs. 86.0±7.8 fold SC; MCT4: RC 2.9±0.4 vs. 54.6±0.6 fold SC; p<0.05). RC enhanced UCN3 gene expression (4.2±0.5 RC vs. 2.2±0.2 fold SC; p<0.05) and increased UCN3/insulin co-staining (80±1.2% RC vs. 30±2.4% SC; p<0.0001) with enhanced GSIS stimulation index (4.1±0.1 RC vs. 2.8±0.1 SC; p<0.0001). Hypoxia-induced expression was reduced in HI RC compared with freshly isolated islets (LDHA: 0.3±0.1 RC vs. 1.8±0.04 fold SC; MCT4: 0.08±0.01 RC vs. 1.1±0.2 fold SC; p<0.05) with increased UCN3 expression (5.9±1.6 RC vs. 3.3±0.9 fold SC; p<0.05) and enhanced GSIS stimulation index (4.1±0.9 RC vs. 3.0±1.2 SC; p<0.05).

Conclusion: Hypoxia in established PI and isolated HI was reduced over 7 days in dynamic culture. This was associated with enhanced β-cell differentiation and function providing a potential approach to maximise optimal functional mass in islet transplantation.

Disclosure

N.A. Al-Jahdhami: None. S.J. Anderson: None. A. Aldibbiat: None. J.A. Shaw: Advisory Panel; Self; Novo Nordisk A/S.
Pancreatic ß-Cell Specific Knockout of Grb10 Improves ß-Cell Function by Enhancing ß-Cell Differentiation and Suppressing ß-Cell Dedifferentiation
Authors: ZHANG JINGJING , CAI ZIXIN , LIU FENG
Published: June 2018

Aim: Defects in β cell differentiation are associated with type 2 diabetes in humans. Understanding the basis for β cell differentiation defects may reveal new strategies for diabetes therapy.

Results: We found that Growth factor receptor binding protein 10 (Grb10) is highly expressed in mouse and human pancreas and islets and that β cell-specific knockout of Grb10 in mice increased β-cell mass and insulin content, enhanced insulin secretion from β-cells, and up-regulated mTOR and insulin signaling in islets. Pancreatic β cell-specific disruption of Grb10 expression also improved glucose tolerance in mice fed with a high fat diet and protected adult mice from STZ-induced β cell death. Pancreas-specific knockout Grb10 promoted endocrine progenitors differentiate into β cells during embryonic development. In addition, knockout Grb10 in islets alleviated STZ or high fat diet-induced β cell dedifferentiation.

Conclusion: We have identified Grb10 as a key regulator of β cells differentiation/dedifferentiation and demonstrated that reducing the expression level of Grb10 has a protective effect on β-cell function. Our findings suggest a potential effective therapeutic treatment of both type 1 and type 2 diabetes.

Disclosure

J. Zhang: None. Z. Cai: None.
Immunosuppressive PLGA TGF-ß1 Microparticles Induce Antigen-Specific T-Cell Tolerance for Enhancing Islet Transplantation Outcomes
Authors: LI YING , FREI ANTHONY , BARASH STEVEN D. , STABLER CHERIE L.
Published: June 2018

Clinical islet transplantation (CIT) is a potential therapy for type 1 diabetes. Allorejection mediated by the host CD4 T cells is a major hurdle for successful CIT, despite potent systemic immunosuppression (SI). To address these issues, we engineered PLGA microparticles (PLGA-MPs) that allow the localized delivery of TGF-β1 for regulatory T cells (TRegs) induction at the transplant site. TGF-β1 is an immunoregulatory agent that promotes the TRegs differentiation from naïve CD4 cells by inducing FoxP3 expression while suppressing inflammatory Th1 and Th2 responses. To date, we have (i) fabricated TGF-β1 loaded PLGA particles by double emulsion (PVA(aq)- CH2Cl2- PVA(aq)) with characterization in size (45±26 μm), TGF-β1 encapsulation efficiency (49.1±0.1%), and release kinetics (durable biphasic release for 14 days); (ii) proved effective TRegs conversion in OVA-OTII antigen-specific murine model (Fig.1); and (iii) shown efficacy for in vivo Treg conversion by MPs in rodent islet allografts, where accepted grafts showed improved insulin production and elevated TRegs. Future studies exploring the competency of PLGA-MPs to provide durable allograft protection in rodent models is on-going. Based on these promising preliminary results, TGF-β1 PLGA-MPs could provide a translational therapeutic strategy to minimize islet graft rejection and the dependency on SI.

Disclosure

Y. Li: None. A. Frei: Employee; Self; Medline Industries, Inc.. S.D. Barash: None. C.L. Stabler: None.
Islet Beta-Cell Specific Deletion of UBL5 Gene Associated with Mitochondrial Stress Leads to Diabetes in Mice and Beta-Cell Impairment/Death
Authors: HARALAMBOUS CHRISTIAN , NTOUMA VIKTORIA
Published: June 2018

Hyperglycaemia has been shown to cause oxidative stress in pancreatic β-cells, leading to activation of stress responses such as the mitochondrial unfolded protein response (UPRmt). Failure of these responses to adapt to or repair damage from stress results in dysfunction and death of β-cells. Ubiquitin-like protein 5 (UBL-5) is a protein known to have a crucial role in the UPRmt in C. elegans and upregulated during the UPRmt mammals.

The aim of this study was to determine whether UBL-5 has a role in maintaining β-cell mass and function through modulating the UPRmt, by generating and characterising tamoxifen-inducible islet β-cell specific UBL-5 knockout mice. Homozygous β-cell UBL-5 knockout mice (UBL5-/-) showed glucose intolerance and lower plasma insulin levels during the OGTT while heterozygous mice (UBL5+/-) showed no difference. UBL5-/- had significantly reduced plasma insulin levels during IVGTT compared to control. Interestingly, UBL5+/- had significantly increased plasma insulin levels during IVGTT. Beta-cell mass was also significantly reduced in UBL5-/-, with most showing signs of frank diabetes (blood glucose >20 mM), polyuria and polydipsia, while UBL5+/- had increased β-cell mass. One week post UBL5 deletion, UBL5-/- mice had significantly increased blood glucose levels and islet cleaved caspase-3 levels compared to controls despite no difference in β-cell mass. In addition islets taken from UBL5-/- mice 1 week post UBL5 gene deletion showed significant decrease in insulin secretion, suggesting that β-cell dysfunction precedes the decrease in β-cell mass. Islets taken from UBL5+/- mice had increased insulin secretion compared to control. Real time PCR data showed a decrease in UPRmt genes (Clpx, Clpp, CHOP, Lonp1, HSP10, HSP70, ATF5) with HSP60 showing increased expression in the UBL5-/- mice one week after gene deletion, while the UBL5+/- mice showed an overall increase in most UPRmt related genes.

Disclosure

C. Haralambous: None. V. Ntouma: None.
Cyclophilin D-Dependent Mitochondrial Proton Leak in ß Cells Promotes Basal Insulin Secretion
Authors: ALSABEEH NOUR , TADDEO EVAN , WIKSTRÖM JAKOB D. , RITOU ELENI , STILES LINSEY , KIBBEY RICHARD , LIESA MARC , SHIRIHAI ORIAN
Published: June 2018

Prediabetic subjects display hypersecretion of insulin at basal levels of blood glucose, a phenomenon termed basal hypersecretion. The molecular mechanisms underlying basal hypersecretion and how it may lead to beta cell failure are not well understood. Islets isolated from prediabetic animals and humans have an inherently high mitochondrial proton leak. However, the molecular entity responsible for this leak and its role in basal hypersecretion are not known. We hypothesize that basal secretion is regulated by proton leak mediated by the permeability transition pore (PTP) functioning in a low conductance state. Our data indicate that islets from high fat diet animals display basal hypersecretion of insulin, increased mitochondrial proton leak, and increased expression of Cyclophilin D, a PTP regulator. Moreover, acute stimulation of islets with amino acids (leucine/glutamine) and fatty acids (oleate/palmitate) at basal glucose concentrations increases leak. Pharmacological and genetic inhibition of Cyclophilin D reverses nutrient induced islet leak and insulin secretion. Pharmacological stimulation of mitochondrial proton leak is sufficient to induce basal hyper secretion. Inhibition of CypD-mediated proton leak might constitute a novel target to test the role of basal hyperinsulinemia in the development of diabetes and obesity.

Disclosure

N. Alsabeeh: None. E. Taddeo: None. J.D. Wikström: None. E. Ritou: None. L. Stiles: None. M. Liesa: None. O. Shirihai: None.
Altered Expression of Insulin Signaling Proteins in Islets from T2DM Female Mice
Authors: SONDGEROTH KORIE L. , LEPARD KATHY
Published: June 2018

With development and progression of T2DM in KKAy mice, pancreatic islets alter their expression of GLUT2, insulin and insulin receptor. The study’s goal was to identify glucoregulatory proteins in the insulin receptor signaling pathway that were early markers of islet dysfunction after 4 weeks of uncontrolled hyperglycemia. Isolated islets were pooled from 2 groups of 12 week old mice: insulin-resistant, euglycemic KK mice and insulin-resistant, hyperglycemic KKAy T2DM mice. Samples were analyzed for 40 proteins using 221 antibodies in an insulin phospho antibody array. After 4 weeks of hyperglycemia in KKAy mice, many proteins were not altered as compared to KK mice (ratio: 0.94-1.02) including insulin receptor (Phospho-Tyr1355), FOXO1A (Phospho-Ser329), PI3-kinase p85-alpha (Phospho-Tyr607), ERK1/2, PP1-alpha, and AMPK1. The phosphorylation states of some proteins were altered including AKT1 (Phosphor-Ser124) (0.77) and IRS-1 (Phosphor-Ser636) (1.25). Three proteins were then selected for ELISA analysis in islets isolated from 4, 8 (onset of T2DM), and 12 week old mice: GLUT2, iNOS (1.41) and CBL (Phospho-Tyr700) (0.78), an E3 ubiquitin ligase. GLUT2 was lower in KKAy mice at 4 weeks (KK, 4.0±0.5; KKAy, 2.3±0.3, p<0.001), but not at 8 or 12 weeks, because GLUT2 decreased with age in both groups (12 weeks: KK, 1.0±0.1; KKAy, 1.4±0.1, p=0.31) [age*mouse: F(2,18)=11.104, p=0.001]. CBL-b was higher in KKAy mice (KK, 0.50±0.09; KKAy, 0.65±0.07) [F(1,17)=4.527, p=0.048] and decreased with age in both groups (12 week: KK, 0.30±0.02; KKAy, 0.45±0.13) [F(2,17)=12.231, p=0.001]. In contrast, iNOS was lower at 4 week (KK, 3.0±0.3; KKAy, 2.0±0.4; p=0.029), then higher at 8 and 12 week (KK, 0.8±0.4; KKAy, 2.1±0.2; p=0.007) with iNOS steady in KKAy mice [age*mouse: F(2,16)=10.902, p=0.001]. With short-term hyperglycemia in KKAy mice, many insulin signaling proteins were not altered as compared to diabetes susceptible KK mice. At onset on T2DM in KKAy mice, sustained iNOS and elevated CBL-b were early markers of islet dysfunction.

Disclosure

K.L. Sondgeroth: None. K. LePard: None.
Hybrid Insulin Peptides Form in Islets of Nondiabetes Prone Mice
Authors: WILES TIMOTHY , HASKINS KATHRYN M. , DELONG THOMAS
Published: June 2018

Using a proteomic strategy, we recently identified a new class of post-translational protein modification that occurs in pancreatic beta cells. This modification is a result of insulin fragments that become covalently linked to other protein fragments that form in beta cells. The resulting hybrid insulin peptides (HIPs) contain non-germline encoded amino acid sequences and provide a plausible explanation on how tolerance to self-antigens may be lost leading to the development of type 1 diabetes (T1D). We demonstrated that autoreactive CD4 T cells, isolated from the residual islets of T1D organ donors, target various HIPs. Additionally, diabetes triggering CD4 T cells from non-obese diabetic (NOD) mice, a major animal model for T1D, were shown to be HIP reactive. Using mass spectrometric analyses on cell extracts of beta cell tumors, isolated from NOD-RIPTAg mice, we confirmed the presence of HIPs in those cells. Key questions that arise from these observations are whether HIP formation is a process that takes place in human beta cells, and whether individuals that do not develop diabetes also form HIPs. BALB/c mice do not develop spontaneous diabetes and here we analyzed pancreatic islets from these mice by mass spectrometry. We verified the presence of various HIPs in those islets. Data indicate that the quantity of HIPs formed in NOD islets is equivalent to the quantity formed in BALB/c islets. However, additional quantification steps will be required to validate these observations. It is difficult to obtain sufficient amounts of residual islets from T1D organ donors that allow a detailed proteomic analysis, but our findings now set the stage to look at more accessible islets of non-T1D organ donors to verify the formation of HIPs in humans.

Disclosure

T. Wiles: None. K.M. Haskins: None. T. Delong: None.
Investigating the Role of CD137 Ligand in the Pathogenesis of Type 1 Diabetes
Authors: FODA BARDEES , FORSBERG MATTHEW H. , CIECKO ASHLEY E. , MUELLER KEVIN W. , GEURTS ARON , CHEN YI-GUANG
Published: June 2018

Pathogenesis of type 1 diabetes (T1D) involves various interactions between genetic and environmental factors. The gene encoding the co-stimulatory molecule CD137 is located within the Idd9.3 T1D susceptibility locus and contributes to diabetes progression in NOD mice. We have previously shown that CD137 expression in T cells has dual functions: CD4+CD137+ T cells negatively regulate T1D development while CD8+CD137+ T cells showed potent diabetogenecity. The protective function of CD137 in CD4+ T cells is likely due to the significant amounts of soluble CD137 (CD137) produced by Foxp3+ Tregs. The interaction between CD137 and its ligand (CD137L) induces two signaling pathways, forward one driven by CD137 and the reverse signaling mediated by CD137L, both of which modulate T cell function. Here, we study the impact of CD137L deficiency on T1D to gain further insight into disease pathogenesis. We successfully generated a mouse strain with the NOD background and knockout of the gene encoding CD137L (Tnfsf9) using CRISPR/Cas9 technology. Relative to wild type NOD, Tnfsf9-/- mice showed significant delay in T1D development, less islet-infiltrating autoreactive CD8 T cells, reduced high-avidity IGRP autoreactive CD8 T cells in the spleen and pancreatic lymph node, and less inflamed islets. Interestingly, we detect significant increase in serum levels of sCD137. Furthermore, we could not detect differences in CD137+ Treg populations between NOD and Tnfsf9-/- mice. In bone marrow transfer experiments, CD137L deficiency in either hosts or donors was able to suppress T1D development. Tnfsf9-/- and wild type T cells showed similar capacity to induce T1D in NOD.Rag1-/- recipients. We are working on identifying the cellular source of increased sCD137 and the mechanism behind this elevation. We direct our efforts to understand how CD137-CD137L interaction can modulate T1D pathogenesis and how to translate the data from NOD mouse to understand the pathogenesis of human diabetes.

Disclosure

B. Foda: None. M.H. Forsberg: None. A.E. Ciecko: None. K.W. Mueller: None. A. Geurts: None. Y. Chen: None.
Acceptance of Murine Islet Allografts without Immunosuppression in the Inguinal Subcutaneous White Adipose Tissue Pretreated with bFGF
Authors: NAKAFUSA YUKI , NITTA NAOYOSHI , NAKAMURA MASAFUNI , YASUNAMI YOHICHI
Published: June 2018

Prevention of immune rejection without immunosuppression is a goal for transplant immunology. Previous report demonstrated that rejection of rat islet allografts in sc space of the back between low responder strains was prevented by pretreatment of the site with bFGF (AJT2014). However, it was not in the high responder combination of mice (BALB/c to C57BL/6). Here, we determined whether islet allografts in the inguinal subcutaneous white adipose tissue (ISWAT), a novel site of islet transplantation (tx), pretreated with bFGF are accepted without immunosuppression in mice. An agarose rod containing bFGF was buried in the ISWAT of a C57BL/6 mouse, and 1-3 weeks later 400 BALB/c islets (2 donors) were grafted in the space after the removal of a rod. Recipients were made diabetic by STZ (180mg/kg, iv) at 3 days prior to tx. Eventually, we found that 10μg bFGF/50μl/rod with 2 week interval is the optimal, in which 11/14 recipient mice remained normoglycemic (<200mg/dl) for more than 60 days. Removal of the ISWAT with islets promptly made recipient mice hyperglycemic again. Histologically, intact islets were seen in the ISWAT. In contrast, islet allografts (n=7) in the ISWAT pretreated with vehicle were rejected by 10 days. FACS revealed an expansion of CD8 T cells in the ISWAT rejecting but not accepting islet allografts. In the latter, IL-10+cells including Gr-1+ cells were seen. Re-tx of BALB/c islets (n=3) to the contralateral ISWAT of normoglycemic mice induced rejection of the initial BALB/c grafts and made recipient mice hyperglycemic again, while that of C3H (n=3) did not. These findings indicate that acceptance of murine islet allografts without immunosuppression was achieved when the ISWAT pretreated with bFGF was the site and that the acceptance appeared mediated by local rather than systemic unresponsiveness.

Disclosure

Y. Nakafusa: None. N. Nitta: None. M. Nakamura: None. Y. Yasunami: None.