bluuebunny/crossref_metadata_2025 huggingface dataset:
The First Genome-Wide Association Study (GWAS) for Type 2 Diabetes in Youth from the Progress in Diabetes Genetics in Youth (ProDiGY) Collaboration
Authors: SRINIVASAN SHYLAJA , TODD JENNIFER , CHEN LING , DIVERS JASMIN , GIDDING SAM , CHERNAUSEK STEVEN , GUBITOSI-KLUG ROSE , KELSEY MEGAN M. , SHAH RACHANA , BLACK MARY HELEN , WAGENKNECHT LYNNE E. , MERCADER JOSE M. , MANNING ALISA , FLANNICK JASON , DABELEA DANA , FLOREZ JOSE C.
Published: June 2018

The prevalence of type 2 diabetes is increasing in youth, particularly in certain ethnic groups, and studies have highlighted differences between youth- and adult-onset disease. However, its genetic determinants remain largely unexplored.

To identify genetic variants predisposing to type 2 diabetes in youth, we formed ProDiGY, a multi-ethnic collaboration of the TODAY, SEARCH for Diabetes in Youth, and T2D-GENES studies, with 30young cases (mean age= 15.1 y ±2.8, 63% female, 22% white, 36% African American, 42% Hispanic) and 6061 diabetes-free adult controls (mean age = 54.2 y ±12.4; 57% female, 24% white, 18% African American, 58% Hispanic).

After stratifying by self-reported and principal component-clustered ethnicity, we performed association analyses on 206,928 directly genotyped variants using a generalized linear mixed model using a genetic relationship matrix to account for population structure.

We identified 5 genome-wide significant loci, including the novel locus rs13130484 in GNPDA2 (P= 1.4 ×10-8, odds ratio [OR]= 1.24), which has nominal association with type 2 diabetes in adults. Known loci identified in our analysis include rs4132670 in the well-known type 2 diabetes locus TCF7L2 (P= 4.2×10-13, OR 1.35) in high linkage disequilibrium with causal SNP rs7903146 (R2=0.83), rs11662368 near the obesity locus MC4R (P= 3.9×10-9, OR =1.31), rs11257655 in RN7SL232P (P= 5.7×10-12, OR= 1.33), and rs7075724 in CDC123 (P= 8.3×10-11, OR= 1.29); effect sizes are greater for these latter three loci than those found in adults (Pdiff= 8.2×10-5, 2.4×10-5, and 1.7×10-6, respectively). Secondary analysis with 856 diabetes-free youth controls demonstrated consistent direction of effect but no additional loci.

In conclusion, we identified known and novel genetic loci predisposing to type 2 diabetes in youth. Our future analysis of ∼10 million variants holds the potential to uncover additional new type 2 diabetes susceptibility loci.

Disclosure

S. Srinivasan: None. J. Todd: None. L. Chen: None. J. Divers: None. S. Gidding: None. S. Chernausek: Advisory Panel; Self; Novo Nordisk Inc.. R. Gubitosi-Klug: None. M.M. Kelsey: Other Relationship; Self; Daiichi Sankyo Company, Limited, Merck Sharp & Dohme Corp.. R. Shah: None. M. Black: None. L.E. Wagenknecht: None. J.M. Mercader: None. A. Manning: None. J. Flannick: None. D. Dabelea: None. J.C. Florez: Consultant; Self; Intarcia Therapeutics, Inc.. Consultant; Spouse/Partner; Santen.
A High Resolution Capture-C Promoter "Interactome" Implicates Causal Genes at Type 2 Diabetes GWAS Loci
Authors: MANDUCHI ELISABETTA , JOHNSON MATTHEW , LEONARD MICHELLE , LU SUMEI , HODGE KENYAITA M. , CHESI ALESSANDRA , WELLS ANDREW D. , GRANT STRUAN F.A.
Published: June 2018

GWAS has revealed many loci for type 2 diabetes (T2D). However, GWAS just reports genomic signals and not necessarily the precise localization of effector genes, with eQTLs making inferences to only a subset of such loci. Chromatin conformation capture-based techniques that detect contacts between distant regions of the genome offer an opportunity to understand GWAS signals that principally reside in non-coding regions, thus likely influencing regulatory elements. To move beyond analyzing one locus at a time and to improve on the low resolution of available Hi-C data, we developed a high resolution Capture-C based method to simultaneously characterize the genome-wide interactions of all human promoters in any cell type. We applied this to the immortalized human β-cell line, EndoC-βH1, a model relevant to T2D. We designed a custom Agilent SureSelect library targeting both ends of DpnII restriction fragments that overlap promoters of protein-coding, plus noncoding, transcripts, totaling 36,691 RNA baited fragments. Following sequencing, we investigated significant interactions at varying resolutions depending on how the fragments were leveraged and/or collapsed. In parallel, we generated ATAC-seq open chromatin maps to filter for informative proxy SNPs (r2>0.8) to each of the 104 T2D independent sentinels reported to date, yielding overall 150 (harbored in 94 DpnII fragments) for 50 of these loci. By filtering our promoter ’interactome’ results at 4 DpnII fragment resolution (median distance between interacting regions ∼115kb, median region size =1,440bp) based on these proxies and openness of the regions they interact with, we observed contacts relevant to 17 of the original loci. Some ’nearest’ genes were supported e.g., GIPR and ZFAND3, while at other loci more distant genes were implicated e.g., OGFOD2 at ’MPHOSPH9’ and MESP2 at ’AP3S2’.

In conclusion, we observed informative contacts at putative effector genes for 16% of T2D GWAS loci in this particular cellular context.

Disclosure

E. Manduchi: None. M. Johnson: None. M. Leonard: None. S. Lu: None. K.M. Hodge: None. A. Chesi: None. A.D. Wells: None. S.F.A. Grant: None.
Genetics of Circulating HbA1c Long-Term Change over Time in Nondiabetic Subjects—The Long Life Family Study
Authors: AN PING , LENZINI PETRA , THYAGARAJAN BHARAT , LEE JOSEPH H. , ZMUDA JOSEPH M. , VARDARAJAN BADRI N. , MILJKOVIC IVA , YASHIN ANATOLIY I. , CHRISTENSEN KAARE , PERLS TOM , PROVINCE MICHAEL
Published: June 2018

HbA1c is a stable index of chronic glycemic status and hyperglycemia associated with progressive development of insulin resistance and frank T2D. Trajectories of repeated HbA1c measurements over years are also associated with aging, cognitive performance and mortality. To identify genomic regions and loci for long term change in HbA1c, we conducted genome-wide linkage (GWLS) and association (GWAS) studies in the Long Life Family Study (LLFS), a study with familial clustering of exceptional longevity in the U.S. and Denmark. We performed GWLS/GWAS in 1902 non-T2D participants of European ancestry who had complete data from two exams collected 7 years apart. HbA1c change over time was derived using growth curve modeling, and was adjusted for age, sex, BMI, smoking, centers and PCs, and blom-transformed to approximate normality. SNPs imputed using the HRC all-Caucasian reference panel were analyzed. LOKI/SOLAR were used for GWLS. EPACTS with make-kin and emmax options for familial clustering assuming additive genetic effects were used for GWAS. Lods > 3 for GWLS and p < 5e-8 for GWAS were used to declare significance. Heritability for HbA1c change over time reached 38% (p = 8e-10). GWLS yielded a significant region on 17p12 (Lods 3.5, 13 Mb). Two previous reports highlighted this region for fasting glucose and metabolic syndrome. From the GWAS, one SNP on 10q22.1 was significant (HK1-rs16926246, p = 4e-8, MAF = 0.14, β = -0.26). HK1 encodes hexokinase 1, which phosphorylates glucose to produce G6P, the first step in glycolysis. HK1-rs16926246 is a SNP previously associated with baseline HbA1c, erythrocyte traits, hematocrit and hemoglobin. Together, we identified 17p12 and HK1-rs16926246 as candidate loci for longitudinal change in HbA1c among non-T2D subjects in the LLFS. This analysis yields new information about the genetic regulation of HbA1c among nondiabetic individuals selected for exceptional survival and healthy aging. Replication in the external datasets is underway.

Disclosure

P. An: None. P. Lenzini: None. B. Thyagarajan: None. J.H. Lee: None. J.M. Zmuda: None. B.N. Vardarajan: None. I. Miljkovic: None. A.I. Yashin: None. K. Christensen: None. T. Perls: None. M. Province: None.
Low Vitamin B12 Levels in Early Pregnancy Are Associated with Fasting Glycemia—A Prospective Cohort Study
Authors: SUKUMAR NITHYA , ANTONYSUNIL ADAIKALA , GHEBREMICHAEL-WELDESELASS YONAS , GOLJAN ILONA , BAGIAS CHRISTOS , SARAVANAN PONNUSAMY
Published: June 2018

Background: The pathogenesis of gestational diabetes mellitus (GDM) is not fully understood and combining novel biomarkers with established risk factors could improve early prediction of GDM. Low vitamin B12 (B12) levels have been linked to obesity, insulin resistance and GDM but its predictive ability in early pregnancy as a risk factor for later GDM has not been proven.

Methods: Micronutrients in Pregnancy as a Risk factor for gestational diabetes and Effects on mother and baby (PRiDE) is a cohort study of pregnant women at high risk of developing GDM (n=4751 recruited). GDM was diagnosed by a 2-hour 75g OGTT according to UK NICE criteria (fasting blood glucose, FBG ≥5.6mmol/l or 2-hour BG ≥7.8mmol/l). B12 and folate were measured by electrochemiluminescence and deficiency defined as ≤200pg/ml and ≤3ng/ml respectively. Multiple linear and logistic regression models were done with covariates including age, BMI, ethnicity, parity, smoking status, gestational weight gain and folate levels.

Results: The data from the first 3412 women with complete B12/folate and OGTT results were analysed (median (range) gestational age 12+5(4+3, 16+0) and 26+4 (15+0, 32+1) weeks respectively). Mean (±SD) age was 30.7±5.2 years, BMI 30.8±7.3 kg/m2 and 56.0% were obese. 11.5% had GDM and B12 and folate deficiency rates were 10.2% and 0% respectively (p>0.05 in GDM vs. no-GDM). GDM women had similar B12 and folate values to no-GDM (346±151 vs. 357 ± 155 pg/ml and 19.8±12.6 vs. 18.8±12.1 ng/ml respectively, adjusted p>0.05). B12 was negatively correlated with BMI (Pearson’s r=-0.24, p<0.001) and inversely related to FBG, after adjustment for covariates including BMI (β=-0.03, p=0.003). There was no association with 2-hour BG. Low B12 values were more predictive of GDM among obese women (mean 314±119 vs. 328±134 pg/ml, adjusted p=0.06).

Conclusions: Early pregnancy B12 values were predictive of high FBG in late gestation. Low B12 levels may be an independent risk factor for GDM, particularly in obese women.

Disclosure

N. Sukumar: None. A. Antonysunil: None. Y. Ghebremichael-Weldeselass: None. I. Goljan: None. C. Bagias: None.
Hepatic Transcriptomic Signature of High-Sucrose Diet in Nonobese Model of Metabolic Syndrome and Prediabetes
Authors: MARKOVÁ IRENA , HÜTTL MARTINA , MALINSKA HANA , SEDA ONDREJ , KAZDOVA LUDMILA
Published: June 2018

Both genetic predisposition and nutrition factors, such as high sucrose diet (HSD), are involved in the development of metabolic syndrome and insulin resistance. HSD can cause glucose intolerance and liver steatosis and exert hypertriglyceridemic effect. The aim of this study was to investigate the liver transcriptome profile under standard diet or HSD (70 kcal% as sucrose) in two strains of rats. We used Wistar rats and a strain of hereditary hypertriglyceridemic rats (HHTg) as a polygenic model of metabolic syndrome and insulin resistance. HSD feeding in 2 weeks did not affect body weight, but fasting glucose and AUC were increased (P diet<0.01, P strain<0.05). HSD feeding in both strains led to marked accumulation of hepatic triglycerides (P diet<0.01). However, cholesterol concentrations in the liver were reduced after HSD (P diet<0.01) in both strains. Comparative transcriptomic analyses in the liver identified (FDR<0.05) sets of differentially expressed genes in response to HSD common to both strains (n=959 transcripts) as well as HHTg (n=687) and Wistar (n=1531) specific sets. Network analysis revealed overrepresentation of genes involved in transport, synthesis or regulation of lipid metabolism (SREBF, PPARA, PPARG, SCD), mitochondrial dysfunction, excessive oxidative stress response (NRF2-mediated) and endoplasmic reticulum stress. Expression of genes related to sterol biosynthesis and oxidative phosphorylation was down-regulated, whereas genes involved in fatty acid synthesis (FAS, ACC), oxidative or endoplasmic reticulum stress were up-regulated after HSD. Our results support that HSD induced alterations in the liver transcriptome in both strains, which precede the development and progression of liver steatosis. The top regulated gene networks included lipid metabolism and mitochondrial dysfunction. Most of the major expression shifts due to HSD were similar in direction in both strains but differed in magnitude of expression change.

Disclosure

I. Marková: None. M. Hüttl: None. H. Malinska: None. O. Seda: None. L. Kazdova: None.
Central Obesity in Early Pregnancy Increased Risk for Gestational Diabetes
Authors: ZHU YEYI , HEDDERSON MONIQUE , QUESENBERRY CHARLES , FENG JUANRAN , FERRARA ASSIAMIRA
Published: June 2018

Central obesity has been linked to type 2 diabetes but prospective data are limited among pregnant women. We examined the associations of central obesity measures, waist-to-hip ratio (WHR) and waist circumference (WC), in early pregnancy with subsequent gestational diabetes (GDM) risk; and evaluated the potential for insulin resistance markers to mediate the central obesity and GDM association. Within the Pregnancy Environment and Lifestyle Study prospective cohort of 1,750 women, waist and hip circumferences were measured at 10-13 weeks of gestation. In a nested case-control study within the cohort, 115 GDM cases ascertained by Carpenter-Coustan criteria and 230 non-GDM controls had fasting serum insulin, homeostasis model assessment of insulin resistance (HOMA-IR), and adiponectin measurements at weeks 16-19. Multivariable Poisson and conditional logistic regression models were used, adjusting for established risk factors including age ≥35 years, minority race/ethnicity, pre-pregnancy overweight/obesity, family history of diabetes, previous GDM, and preexisting hypertension. Compared to women with WHR <0.85 and no established risk factors, women with WHR ≥0.85 and no risk factors had a 3.74-fold (95% CI 1.92-7.27) increased risk of GDM, and women with WHR ≥0.85 and at least one risk factor had a 6.63-fold (3.48-12.63) increased risk. In the receiver-operating-characteristic curve analysis, WHR significantly improved GDM risk prediction beyond established risk factors (C-statistics 0.796 vs. 0.744, P-for-difference <0.0001). Similar but attenuated results were observed for WC ≥88cm. Insulin, HOMA-IR, and adiponectin levels mediated the WHR-GDM association by 9.0%, 9.6%, and 11.1%, respectively; corresponding mediation proportions for WC-GDM were 40.0%, 41.1%, and 35.4% (all P-values <0.04). Our findings suggest that central obesity in early pregnancy represented a high-risk phenotype for GDM and may help identify at-risk women for early screening, prevention, and treatment.

Disclosure

Y. Zhu: None. M. Hedderson: None. C. Quesenberry: None. J. Feng: None. A. Ferrara: None.
Renal C3 Production Drives Acute Kidney Injury and Acute Kidney Disease in Diabetics with Sepsis
Authors: FRYDRYCH LYNN M. , BIAN GUOWU , WARD PETER A. , BITZER MARKUS , DELANO MATTHEW
Published: June 2018

Introduction: Type 2 diabetics (T2D) suffer the greatest sepsis mortality due to the development of acute kidney injury (AKI), which progresses to acute kidney disease (AKD), renal failure, and death. Systemic inflammation and complement production are known to provoke AKI during sepsis. We hypothesize that sepsis induced renal inflammation and complement production drives progression of AKI to AKD in T2D.

Methods: C3-/-, TLR2-/-, TNF-/-, C57BL/6, and Diet Induced Obese (T2D) 30 week old mice underwent sepsis from cecal ligation and puncture (CLPLD20) or sham procedure. At serial time points, plasma and kidneys were harvested (n=5 mice/group). Multiplex RT-PCR arrays were used to evaluate toll like receptors (TLR), complement, and immune signaling cascades. TNF, BUN, creatinine, and NGAL were evaluated by ELISA.

Results: In lean mice, renal NGAL, representing AKI, peaked at 1 day and returned to baseline 7 days after CLP. In T2D mice, NGAL persisted past 7 days and peaked after 14 days, indicating transition to AKD (p<0.001). Renal C3 mRNA levels were eight-fold higher 1 day after CLP in lean and T2D mice compared to sham (p<0.001). In lean mice, C3 levels returned to baseline at 7 days. C3 levels in T2D mice peaked at 2 weeks and persisted. Renal in-situ hybridization showed significantly more C3 in septic T2D mice compared to lean mice. RT-PCR analysis showed significantly more renal TLR2, TNF, and C3 mRNA in T2D mice compared to lean mice. C3, TLR2, and TNF null mice each produced less renal C3 and NGAL, demonstrating less AKI and AKD (p<0.001). In T2D mice, TNF inhibition blocked C3 and NGAL production, representing prevention of AKI and AKD after sepsis.

Conclusion: Lean mice resolve sepsis induced AKI by 7 days. In T2D mice, AKI persists past 7 days and progresses to AKD. The T2D AKI to AKD progression is dependent on local renal C3 production through TLR2 and TNF signaling. TNF inhibition is a novel strategy to prevent AKI progression to AKD, preserve renal function, and improve T2D sepsis mortality.

Disclosure

L.M. Frydrych: None. G. Bian: None. P.A. Ward: None. M. Bitzer: None. M. Delano: None.
Phosphoregulation Profiles of Transcription Factors during Early Stage of Thermogenesis in Brown Fat
Authors: GAO XIAOJING
Published: June 2018

Brown adipose receives extensive attention for dissipating stored chemical energy in heat via nonshivering thermogenesis. However, whether and how the dynamics of key transcription factors (TFs) phosphorylation contributes to initial activation of thermogenesis remains unknown. Here, we employed the stat-of-the-art data-independent acquisition (DIA) mass spectrometry to explore the global and temporal phosphorylation profiles of signaling networks to elucidate mechanism responsible for the TF activation for thermogenesis gene transcription. The nucleus was isolated before and after stimulation for 5, 10, 20, 30, 40, 45, 50, 55, 60, 70 and 80 min with NE. Consequently, 401 transcription regulators bearing 1007 phosphosites were identified with high reproducibility across 12 time points. Firstly, the UCP1 transcriptional level was measured, which began at 30min, then rose until 70min to the peakedness. The global phosphoproteome displayed dramatic changes early to 20-30min, which could be classified into two dynamic clusters, namely UCP1 pre-transcription phase and transcription phase. The former changed from 5min, and reached the extremum at 20-40min, which functioned before UCP1 action. Some well- characterized transcriptional regulators of UCP1 were founded to be phosphorylated, such as PRDM16 and RIP140. Alternatively, other phosphorylation events of TFs such as Jun and ATF2 began from 30min, and maintained until 70min, almost synchronizing with UCP1 transcription. Intriguingly, we found THRAP3 posed multiple phosphosites but involving in different regulation patterns. This might indicate that THRAP3 had distinct transcriptional regulation and functions upon thermogenesis. We, for the first time, uncovered temporal phosphoregulation profiles of TFs, which might be potentially required for initial activation of thermogenesis in brown fat. A new sight was provided to increase energy expenditure for treatment of obesity and other metabolic syndrome.

Disclosure

X. Gao: None.
Fat Shadows from DEXA for Documentation of Fat Distribution in Patients with Lipodystrophy
Authors: MERAL RASIMCAN , RYAN BENJAMIN J. , HOROWITZ JEFFREY F. , ORAL ELIF A.
Published: June 2018

Lipodystrophy (LD) syndromes are a heterogeneous group of disorders causing atypical diabetes, associated with selective absence of fat with diagnosis mostly depending on the clinical acumen of the physician. With niche therapies either approved or under investigation, development of objective diagnostic tools for these syndromes are urgently needed. Here we describe a new method using the built-in features of the enCore software v14.10 to render out non-fat tissues from Dual Energy X-ray Absorptiometry (DEXA) scans to derive a “fat-shadow” (Figure). We evaluated fat-shadows from 58 LD (50F:8M) patients and 91 non-LD patients (51F:40M). Among the LD, 3 had Generalized LD, 2 had Acquired Partial LD, and 53 had Familial Partial LD. Controls consisted of a mixed population of lean (12F:14M) or obese patients (39F:26M) with or without metabolic disease. There was substantial overlap in % body fat between the LD and non-LD groups (interquartile range: total, 22.2-39.8% vs. 33.6-45.9%; trunk, 27.0-46.8% vs. 39.1-50.2%; legs, 16.6-30.6% vs. 27.7-42.6%). However, fat-shadows obtained from patients with LD stood out due to the paucity of fat signal either in a generalized or regional manner compared to controls. Overall, fat-shadows provided sufficient qualitative information to infer clinical phenotype. We propose that these fat-shadows could be used for accurate documentation of fat distribution in LD.

Disclosure

R. Meral: None. B.J. Ryan: None. J.F. Horowitz: None. E.A. Oral: Advisory Panel; Self; Aegerion Pharmaceuticals. Research Support; Self; Aegerion Pharmaceuticals, Akcea Therapeutics. Advisory Panel; Self; Akcea Therapeutics. Research Support; Self; Ionis Pharmaceuticals, Inc., AstraZeneca. Other Relationship; Self; Aegerion Pharmaceuticals.
National Trends in Type 2 Diabetes Treatment—Comparing Older and Younger Adults
Authors: PILLA SCOTT J. , SEGAL JODI B. , CALEB ALEXANDER G. , BOYD CYNTHIA , MARUTHUR NISA
Published: June 2018

With expanding therapeutic options for type 2 diabetes, the treatment of older adults requires special care to balance safety and complexity with glucose control. We compared national trends in type 2 diabetes treatment among older (≥65 years) and younger adults (30-64 years) using the 2006-2015 National Ambulatory Medical Care Survey (NAMCS), an annual probability sample of visits to U.S. outpatient providers. We included all visits of patients with type 2 diabetes using ≥1 diabetes medication. Analyses were weighted to yield nationally-representative estimates. In the most recently available data, 2014-2015, there were 23.3 million and 20.1 million annual treated diabetes visits for older and younger adults, respectively. The most frequently used medications in older and younger adult visits were metformin (50.4% vs. 62.3% respectively, p<0.001), sulfonylureas (31.1% vs. 26.7%, p=0.301), and long-acting insulins (30.2% vs. 22.4%, p=0.017). GLP-1 receptor agonists were used in fewer older than younger adult visits (2.9% vs. 6.2%, p=0.004); DPP-4 inhibitor use was similar (12.9% vs. 11.1%). In the past decade, mean diabetes medication counts increased in both older and younger adult visits (1.47 to 1.54, p-trend=0.046; and 1.53 to 1.67, p-trend 0.052, respectively). Between 2006 and 2015, the proportion of visits with metformin, DPP-4 inhibitors, and insulin increased for both older and younger adults; sulfonylurea and thiazolidinedione use decreased (p<0.05 for all trends). Long-acting insulin use increased markedly in older adults, particularly between 2010-2015 where it rose from 12.5% to 30.2% of older adult visits. In younger adult visits, the increase was modest, from 17.2% to 22.4%, a slower rate of increase than in older adults (p-interaction <0.001).

In conclusion, the outpatient treatment of type 2 diabetes differs between older and younger adults driven in part by a marked increase in the use of long-acting insulin among older adults, which may have implications for risk of hypoglycemia.

Disclosure

S.J. Pilla: None. J.B. Segal: None. G. Alexander: Other Relationship; Self; See disclosure. C. Boyd: None. N. Maruthur: None.
Multitrait Genome-Wide Association Analysis of Macronutrient Intake Identifies 12 Novel Loci for Dietary Intake and Unravels Genetic Overlap with Lifestyle Traits and Cardiometabolic Diseases
Authors: DASHTI HASSAN S. , MERINO JORDI , LANE JACQUELINE M. , FLOREZ JOSE C. , RUTTER MARTIN K. , SAXENA RICHA
Published: June 2018

Suboptimal diets are associated with increased risk of type 2 diabetes, cardiovascular disease and cancer. Genome-wide association studies (GWAS) for individual macronutrients have facilitated the discovery of two relevant loci (FGF-21, FTO), however a single-nutrient approach may preclude the discovery of additional relevant loci. Here, we conducted a multi-trait macronutrient GWAS for carbohydrate, fat, and protein in the UK Biobank to factor in the significant correlation among macronutrient intake. In the UK Biobank, dietary data were collected from over 210,000 individuals aged 37-74 using the Oxford WebQ, a web-based 24 hour diet recall. For multi-trait GWAS, we combined GWAS association evidence of ∼11m imputed variants in participants of European ancestry (n =192,005) for percent of carbohydrate, fat, and protein adjusted for age and sex by applying the CPASSOC package. We identified 14 independent genome-wide significant (Pυ x 10-8) loci for macronutrient intake. We discovered loci associated with lipid metabolism (APOE, GCKR, SLC39A8, PPP1R3B, MLXIPL), alcohol intake (ADH1B, KLB), and new candidate genes (CCDC171, PSMD7, ARL17B, METTL4), and replicated previously associated loci with dietary intake (FGF-21 and FTO). Of the 14 signals, 12 replicated (P003C;0.0036) in multi-trait analyses using previous GWAS on dietary intake from the CHARGE Consortium (n =91,114). Tissue enrichment analysis across 53 tissues in GTEx revealed enrichment of gene expression in the cerebellum. Genome-wide genetic correlations across 234 traits with publicly available GWAS data indicated shared biological links between dietary intake and lifestyle factors (smoking and education) as well as type 2 diabetes and coronary artery disease (P<2.14x10-4). The present findings expand our understanding of the genetic architecture of dietary intake and the shared genetic links with related traits.

Disclosure

H.S. Dashti: None. J. Merino: None. J.M. Lane: None. J.C. Florez: Consultant; Self; Intarcia Therapeutics, Inc.. Consultant; Spouse/Partner; Santen. M.K. Rutter: Consultant; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Novo Nordisk Inc.. Consultant; Self; Ascensia Diabetes Care, Roche Diabetes Care Health and Digital Solutions. R. Saxena: None.
DBC1, Deleted in Breast Cancer 1, Is a Nuclear STAT5A-Interacting Protein in Adipocytes, but Its Primary Effects on GLUT4 Gene Expression and Lipolysis Are STAT5 Independent
Authors: ABLE ASHLEY , RICHARD ALLISON J. , STEPHENS JACQUELINE M.
Published: June 2018

STAT5A (signal transducer and activator of transcription 5A) is a transcription factor that plays a role in adipocyte development and function. Although the role of STAT5A in fat cell development is well characterized, its specific roles in mature fat cells are still largely unknown. Moreover, the potential role of STAT5A in metabolic diseases, in particular type 2 diabetes, remains unclear. To further understand the function of STAT5A in adipocytes, we employed a non-biased co-immunoprecipitation and mass-spectrometry-based approach to identify novel STAT5A-interacting proteins. One of the proteins we identified was DBC1 (deleted in breast cancer 1; also known as CCAR2). DBC1 is typically localized in the nucleus and has been primarily studied in tumor cells. However, the functions of DBC1 in adipocytes are relatively unknown. Using mouse fat cells, we confirmed that there is a physical association between endogenous STAT5A and DBC1 proteins under physiological conditions in the nucleus that is not dependent upon STAT5 tyrosine phosphorylation. Knockdown of DBC1 in 3T3-L1 adipocytes using siRNA did not affect the expression of several STAT5A target genes including socs3, cish, bcl-6, socs2, and igf-1. However, we did observe increased levels glycerol and free fatty acids released from adipocytes with reduced DBC1 following TNF (tumor necrosis factor) α stimulation. Although loss of DBC1 did not alter STAT5 transcriptional activity for the genes examined, it appears to modulate TNFα-mediated lipolysis in a STAT5-independent manner. In addition, DBC1 knockdown increased GLUT4 expression in murine fat cells but did not have a profound effect on TNFα-mediated changes in gene expression. On-going studies are being performed to elucidate the function of the DBC1/STAT5A interaction and to clarify the impact of DBC1 on lipolysis in adipocytes.

Disclosure

A. Able: None. A.J. Richard: None. J.M. Stephens: None.
Adipocyte Gamma-Secretase Inhibition May Alter IL-6 Expression and Trafficking
Authors: SPARLING DAVID , MCCULLOUGH NILE
Published: June 2018

Adipocyte-mediated inflammatory signaling has been proposed to alter adipose physiology in obesity. Activation of adipocytes can increase secretion of inflammatory cytokines such as IL-6. IL-6 secretion is regulated through changes in expression of both IL-6 itself, as well as its specific SNARE trafficking machinery. Recent data suggests that the γ-secretase enzyme complex is involved in both adipose insulin sensitivity and immune system regulation. We therefore hypothesized that adipocyte IL-6 secretion could be altered by γ-secretase inhibition. To explore this hypothesis, 3T3-L1 adipocytes were stimulated with lipopolysaccharide (LPS) in the absence or presence of a γ-secretase inhibitor. Changes in gene expression of inflammatory cytokines and SNARE complex components, including IL-6, MCP1, Stx6, Vti1b, SCAMP5, VAMP3, Stx4, and SNAP23 were examined by qRT-PCR, and immunofluorescence (IF) was used to determine adipocyte IL-6 expression and localization. IL-6 secretion was analyzed by ELISA. Transcription of IL-6 and several of the SNARE complex proteins were not significantly affected by γ-secretase inhibition. However, total cellular IL-6 increased by LPS stimulation by IF, which could be blocked by pre-treatment with a γ-secretase inhibitor. IL-6-associated SNARE component Vti1b mRNA levels were specifically affected by overnight γ-secretase inhibition. LPS stimulation increased Vti1b transcription 3-fold, which was completely blocked by γ-secretase inhibition (p<0.01, n=6 per condition). Increasing amounts of LPS (range 0 to 10 ng/mL) for 6 hours led to an expected increase in IL-6 secretion, which was significantly blocked by overnight pre-treatment with a γ-secretase inhibitor. LPS-stimulated IL-6 secretion could also be decreased by short-term (30 minute) γ-secretase blockade. Taken together, this data suggests that the γ-secretase enzyme complex may be involved in a variety of signaling cascades controlling both the production and secretion of IL-6 in adipocytes.

Disclosure

D. Sparling: None. N. McCullough: None.
Insulin Receptor–Mediated Signaling Regulates Pluripotency Markers and Lineage Differentiation
Authors: GUPTA MANOJ K. , DE JESUS DARIO F. , KAHRAMAN SEVIM , SHAMSI FARNAZ , HU JIANG , TSENG YU-HUA , KULKARNI ROHIT
Published: June 2018

Insulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells, however its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. To this end, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors (Oct4, Sox2, Klf4 and cMyc). iPSCs were maintained in a 2-inhibitor (2i) feeder-free system. Interestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3 and cMyc were upregulated while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold respectively in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, pmTor and pErk, and an increase in the total mTor and Erk proteins in IRKO iPSCs. Stimulation with leukemia inhibitory factor (LIF) showed a 3-fold decrease of phospho-ERK in IRKO iPSCs (p<0.vs. C, n=3 clones/group). On the contrary, Erk phosphorylation was increased during in-vitro spontaneous differentiation of iPSCs lacking IR. Lineage specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Asci and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Pparg, Fabp4, C/ebpa and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1 and Sox9) (p<0.05, n=3 clones/group). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network and several key proteins involved in various aspects of growth and embryonic development ((p<0.05, n=3 clones/group). Thus, IRKO iPSCs provide an opportunity to explore the crosstalk of insulin receptor signaling with key signaling pathways required for the maintenance of pluripotency and lineage determination.

Disclosure

M.K. Gupta: None. D.F. De Jesus: None. S. Kahraman: None. F. Shamsi: None. J. Hu: None. Y. Tseng: Other Relationship; Self; Chugai Pharmaceutical Co., Ltd.. Research Support; Self; MedImmune. R. Kulkarni: None.
Use of Oral Contraceptives at Child-Bearing Age Are Associated with the Prevalence of Diabetes in Postmenopausal Women
Authors: KIM SUNG-WOO , KIM REBECCA , JEON EONJU , LEE JI-HYUN , SHON HO-SANG
Published: June 2018

Objective: The effect of past use of oral contraceptives (OCs) at child-bearing on the incidence of diabetes and insulin resistance (IR) after menopause is not clearly elucidated. This study aimed to evaluate the association of past use of OCs with the development of diabetes and IR in post-menopausal women.

Methods: This cross-sectional study was based on data from the Korea National Health and Nutrition Examination Survey from 2007 to 2012. Of the 50,4participants, 6554 post-menopausal women were selected and included in the analysis. The long-term effects of OCs use on the prevalence of diabetes in post-menopausal women were examined using multivariate logistic analysis. In addition, fasting glucose and insulin levels were measured in 3338 nondiabetic post-menopausal women, and the association between IR and OCs was examined by analysis of covariance.

Results: The prevalence of diabetes was significantly higher in post-menopausal participants who had taken OCs for more than 6 months than in those who had never taken OCs. The association remained significant after adjusting for multiple confounding factors (odd ratio, 1.379; 95% CI, 1.115-1.707; P=0.003). The duration of OCs use was also positively associated with the prevalence of diabetes. Furthermore, taking OCs for more than 6 months led to a significant increase in fasting insulin levels and HOMA-IR in nondiabetic participants.

Conclusion: Past use of OCs for more than 6 months led to a significant increase in the prevalence of diabetes in post-menopausal women, and increased IR in nondiabetes participants. These results suggested that prolonged use of OCs at a reproductive age is an important risk factor for developing diabetes in post-menopausal women.

Disclosure

S. Kim: None. R. Kim: None. E. Jeon: None. J. Lee: None. H. Shon: None.
An Oral Glucose Load Decreases Post-Prandial Microparticles in Obese Adults With and Without Prediabetes
Authors: EICHNER NATALIE , GILBERTSON NICOLE M. , MUSANTE LUCA , SALVIA SABRINA LA , BARRETT EUGENE , WELTMAN ARTHUR , ERDBRUEGGER UTA , MALIN STEVEN K.
Published: June 2018

Microparticles (MPs) are a novel mediator and/or biomarker of cardiovascular disease (CVD) and type 2 diabetes. A high fat meal can elevate MP release in relation to CVD risk, but the effects of carbohydrates are unknown. This is clinically relevant as low-fat diets are advised for glycemic control. We tested the hypothesis that a 75g oral glucose tolerance test (OGTT) would promote changes in MPs linked to CVD risk. Twenty-five obese adults (Age: 52.4±3.2y, BMI: 32.5±1.2kg/m2) were screened for prediabetes using ADA criteria (75g OGTT and/or HbA1c). Eight were normal glucose tolerant (NGT) and 17 had prediabetes (PD). Body composition (bioelectrical impedance) was measured and arterial stiffness (augmentation index; AI), insulin and glucose were collected at 0 and 2 hour of the OGTT to assess CVD risk. Annexin V+ (AV+) and Annexin V- (AV-) total MPs and platelet MPs (CD31+/CD41+; CD41+) were also collected at these times and analyzed from fresh plasma via imaging flow cytometry. There were no statistical differences in age, BMI, or body fat (all P>0.21) between NGT and PD, although fasting and 2 hour glucose as well as insulin were higher in PD vs. NGT (P<0.03). Glucose and insulin increased during the OGTT (both P≤0.005), while arterial stiffness at 2 hour decreased by 6.9% (P=0.06). Fasting MPs were not different between groups. Total MPs, AV+ CD41+ and AV+ CD31+/CD41+ MPs decreased after the OGTT (P≤0.04), while AV- CD41+ (P=0.08) and CD31+/CD41+ (P=0.10) trended. Attenuated OGTT insulin responses were related to lower post-prandial platelet AV- CD31+/CD41- (r=0.46, P=0.06) and AV- CD41+ (r=0.45, P=0.06) MPs, while arterial stiffness was associated with reduced total MPs (r=-0.49, P=0.03) and AV+CD41+ (r=-0.52, P=0.02). An oral glucose load lowers post-prandial total and platelet MPs in obese adults with NGT and PD. Further work is required to examine MP content in order to gain mechanistic insight for optimizing type 2 diabetes management.

Disclosure

N. Eichner: None. N.M. Gilbertson: None. L. Musante: None. S. La Salvia: None. E. Barrett: None. A. Weltman: None. U. Erdbruegger: None. S.K. Malin: None.
Generation of a Novel Gene Therapy Vector Encoding Beta-Cell–Specific Insulin for Diabetes Treatment
Authors: SAHIN ELIF O. , ERENDOR FULYA , BALCI MUSTAFA KEMAL , SANLIOGLU SALIH
Published: June 2018

Insulin injection is the main treatment modality especially for patients with type 1 diabetes. Despite the development of several insulin analogs, recent meta-analysis indicated that rapid and long acting insulin analogs provide little benefit compared to conventional insulins regarding to glycemic control. Therefore, gene therapy methods are investigated to develop a novel treatment approach to reconstitute a natural endogenous insulin expression profile in diabetic patients. For this purpose, Multisite Gateway Technology was employed to generate a transfer vector carrying lentiviral backbone with minimal insulin promotor hooked up to proinsulin gene sequence. Following confirmation of the transfer plasmid by restriction enzyme digestion and DNA sequence analysis, high-titer LentiINS vectors at a concentration of 10e9 TU/ml were successfully produced by CaPO4 cotransfection of 293T cells with packaging and transfer plasmids. While transduction of NIT1 mouse pancreatic beta cell lines produced 3 fold increase in insulin gene expression compared to controls, no insulin expression was detected in 293T kidney cell line. These results indicate that we successfully constructed an HIV-based lentiviral gene therapy vector being capable of beta cell-specific insulin gene expression and secretion (TUBITAK 215S820).

Disclosure

E.O. Sahin: None. F. Erendor: None. M. Balci: None. S. Sanlioglu: None.
Aquaporins-1 and -3 Play Novel, Distinct Roles in Normal Balanced Insulin Signaling
Authors: WU XIANGDONG , WILLIAMS KEVIN JON
Published: June 2018

Patients with overnutrition, obesity, the atherometabolic syndrome, and type 2 diabetes exhibit imbalanced insulin action, also called pathway-selective insulin resistance. To control glycemia, they require hyperinsulinemia that then overdrives ERK and hepatic de-novo lipogenesis (DNL).

We discovered an oxide transport chain that is required for balanced insulin action (Atherosclerosis 2016;247:225). The chain, abbreviated “NSAPP”, begins when insulin stimulates NOX4 to generate O2•-. NOX4 hands O2•- to SOD3 for conversion into H2O2. Aquaporins (AQPs) channel this H2O2 to inactivate PTEN. Disruption of any component, from NOX4 up to PTEN, leaves PTEN persistently active, thereby producing the same deadly pattern of imbalanced insulin action seen clinically.

We now sought to identify which aquaporins mediate balanced insulin signaling in hepatocytes.

Four AQPs had been reported to act as pores for H2O2: AQP1, 3, 8, and 9. We found that global inhibition of all APQs with low-dose AgNO3 recapitulated the clinical pattern of imbalanced insulin signaling, i.e., full two-site phosphorylation of ERK (pT202/Y204-ERK); monophosphorylation of AKT (pT308-AKT) with only weak phosphorylation at Ser473; robust phosphorylation of AKT targets leading to DNL; but impaired AKT-mediated phosphorylation of FOXO1 (needed to suppress hepatic gluconeogenesis).

Our knockdown of AQP3 recapitulated this pattern of imbalanced insulin signaling. Knockdown of AQP1 produced the mirror-image: strongly inhibited pT202/Y204-ERK and pT308-AKT, but intact pS473-AKT. Knockdowns of AQP8 or AQP9 did not affect insulin signaling to either site on ERK or AKT. Importantly, immunoprecipitation of AQP3 brought down PTEN, a previously unknown partner of any aquaporin.

Thus, AQP3 participates in the NSAPP oxide transport chain. AQP1 plays an unexpected role, perhaps as a scaffold. Our findings will facilitate unraveling the molecular basis for NSAPP dysfunction in overnutrition and hence imbalanced insulin action.

Disclosure

X. Wu: None. K. Williams: Stock/Shareholder; Self; Hygieia, Gemphire, Inc.. Advisory Panel; Self; Gemphire, Inc..
Dissection of Insulin-Dependent Pathways in Skeletal Muscle under Physiological and Diabetic Conditions
Authors: BATISTA THIAGO M. , MARTIN RUBEN GARCIA , CAI WEIKANG , SUK SUJIN , NOH HYE LIM , KIM JASON K. , RONALD KAHN C.
Published: June 2018

Insulin signaling regulates growth and metabolism, in part, by regulation of gene expression. To gain more insight into transcriptional pathways activated by physiological levels of insulin in vivo we performed euglycemic insulin clamp in mice at low (4 mU.kg-1.min-1) and high (12 mU.kg-1.min-1) concentrations of insulin and analyzed changes in gene expression in skeletal muscle by RNA-sequencing. At 3 h, low insulin regulated 451 genes while high insulin regulated 1771 genes by at least 1.5-fold (FDR<0.25). Interestingly, the most up regulated pathways in muscle were potential targets of estrogen-related receptor alpha (Esrra) and were related to TCA cycle and oxidative phosphorylation, while the most suppressed genes were targets of FoxO1 involved in autophagy. Insulin also inhibited the expression of over 40 genes involved in mRNA processing/splicing, mostly within the U2 ribonucleoprotein complex, which regulates the assembly and catalytic activity of the spliceosome. To determine how these insulin-dependent pathways might be altered in diabetes, we intersected the clamp gene expression data with gene expression data in streptozotocin (STZ) or high fat diet (HFD) obesity-induced diabetes focusing on genes oppositely regulated by insulin and diabetes. This approach revealed 93 insulin-regulated genes that were dysregulated in STZ and HFD muscle. Most related to TCA cycle and oxidative phosphorylation, however, changes in mRNAs involved in RNA processing/splicing also overlapped in HFD samples, thus indicating potential insulin resistance-specific gene expression signatures. Together these data demonstrate that insulin is a potent regulator of gene expression in vivo altering mRNA diversity through both changes in expression and dysregulation of the spliceosome, with the latter being most prominent in states of insulin resistance. These differences provide unique targets for therapeutic approaches to type 1 and type 2 diabetes involving muscle.

Disclosure

T.M. Batista: None. R. Garcia Martin: None. W. Cai: None. S. Suk: None. H. Noh: None. J.K. Kim: None. C. Kahn: Advisory Panel; Self; CohBar, ERX Therapeutics, AntriaBio, Inc.. Board Member; Self; Kaleio Biosciences.
Adipocyte Overexpression of p55alpha Subunit of Pi3k Improves Systemic Glucose Metabolism in Lean Mice
Authors: CLAYTON ZACHARY S. , MCCURDY CARRIE
Published: June 2018

Phosphatidylinositol 3-kinase (PI3K), a heterodimeric enzyme composed of a regulatory subunit (p85α/β, p55α/γ, p50α) and a catalytic subunit (p110α/β), is an essential component of the canonical insulin signaling cascade in adipose tissue. Insulin-stimulated PI3K activity is decreased in obese adipose tissue in parallel with a 4-fold increase in p55α abundance; heterozygous deletion of Pik3r1 limits regulatory subunit expression and enhances adipocyte insulin signaling. Additionally, genetic deletion of p55α/p50α in mice has been shown to increase adipocyte glucose uptake and systemic insulin sensitivity. Therefore, we hypothesized that adipocyte-specific p55α overexpression (OX) would reduce adipocyte insulin signaling and decrease systemic glucose tolerance. To test this hypothesis, we generated mice with p55α knock-in at the endogenous ROSA26 locus. The recombination cassette was made by flanking the p55α cDNA and a floxed 4x SV40 polyA STOP signal with FRT and F3. The insertion of Pik3r1 at the ROSA26 locus mediated by Flp-recombinase in ES cells targeted with FRT-PGK-neo-F3 at the ROSA26 locus. The polyA STOP signal was removed by crossing the p55α flox (Fl/Fl) mice with mice carrying Adipoq-cre recombinase (OX). At 12 weeks of age, the metabolic phenotype, glucose tolerance and adipocyte insulin signaling were measured in Fl/FL and OX mice. In adipose tissue, p55α abundance was 7-fold higher in OX compared to FL/FL controls with no difference detected in p85α abundance. Body weight and adiposity was not different between FL/FL and OX mice. Food intake, energy expenditure and the respiratory quotient were not different in OX vs. FL/FL, despite a decrease in total activity in OX mice. Contrary to our hypothesis, glucose AUC calculated from oral glucose tolerance tests was 40% lower (P=0.005) in OX vs. FL/FL. Overall these data suggest that increased expression of the p55α regulatory subunit, in contrast to p85α, enhances PI3K activity and insulin signaling in adipocytes.

Disclosure

Z.S. Clayton: None. C. McCurdy: None.
MEDI0382, a GLP-1/Glucagon Receptor Dual Agonist, Enhances Leptin-Mediated pSTAT3 Induction in Key Neuronal Regions That Regulate Metabolism
Authors: JOUIHAN HANI , BARKHOLT PERNILLE , GRÔNLUND RIKKE , TREVASKIS JAMES
Published: June 2018

MEDI0382 is a potent GLP-1 and glucagon receptor dual agonist peptide that decreases body weight of obese mice by reducing food intake and enhancing energy expenditure. Here we explored whether MEDI0382 may, in part, mediate its effects on metabolism by enhancing leptin action. Diet-induced obese mice were administered vehicle, MEDI0382 (10 nmol/kg; s.c., q.d.) or vehicle and pair-fed to MEDI0382-treated mice for 10 days (n=15-16/group). During this period MEDI0382 induced 11% body weight loss (p<0.01 vs. vehicle), whereas pair-fed mice lost a similar amount of body weight as MEDI0382 group for first 5 days, but rebounded such that final weight loss was 3% from baseline (p<0.vs. vehicle and MEDI0382). On day 10, half of each group was administered vehicle (PBS) or murine leptin (10 mg/kg) and the mice sacrificed 45 min later. Leptin administration increased circulating leptin levels 20-25-fold above endogenous levels. Levels of phosphorylated STAT3 (pSTAT3), a key marker of leptin signaling, were determined in discrete nuclei of the hypothalamus known to regulate energy homeostasis. Within the arcuate nucleus of the hypothalamus pSTAT3 levels were not significantly stimulated by leptin in vehicle or pair-fed mice (indicative of leptin resistance), but were increased in MEDI0382-treated animals. Furthermore, in the dorsomedial, ventromedial and lateral hypothalamic nuclei leptin was able to stimulate pSTAT3 in vehicle and pair-fed mice, but in MEDI0382-treated mice pSTAT3 levels were even further enhanced (p<0.vs. other leptin-treated groups). While we cannot exclude weight loss per se as a factor, these data support the notion that MEDI0382 may mediate its effects on body weight and glycemic control via enhancing central leptin action.

Disclosure

H. Jouihan: None. P. Barkholt: Employee; Self; Gubra. R. Grônlund: None. J. Trevaskis: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca.