bluuebunny/crossref_metadata_2025 huggingface dataset:
246-LB: Rare Noncoding Intronic HNF1A Sequence Variants Cause Aberrant mRNA Processing in MODY
Authors: SOLHEIM MARIE H. , KHAKIANI MOJDEH NASROLLAHZADEH , MOLNES JANNE , AUKRUST INGVILD , NJøLSTAD PåL RASMUS , JOHANSSON STEFAN
Published: June 2019

The transcription factor Hepatocyte nuclear factor-1α (HNF1A) is important for normal pancreas development and function. Some HNF1A genetic variants are associated with type 2 diabetes, others can cause Maturity-Onset Diabetes of the Young (MODY3). Since variations in the HNF1A gene give different clinical consequences, novel sequence variants require thorough clinical and functional analysis. Here we investigate, in vitro, the consequence of rare non-coding intronic sequence variants of HNF1A from the Norwegian MODY registry. To evaluate the effect of four potential splice site mutations, fragments of intron/exon/intron genomic DNA from patient blood was amplified and cloned between two exons in a pCAS2 splicing minigene vector. Total RNA was isolated from HeLa and HepG2 cells transfected with minigene plasmids (wt or mutant) and analyzed by RT-PCR followed by agarose gels and sanger sequencing. All selected variants showed alternative mRNA splicing compared to wt by the minigene assay. The major splicing event of mutant c.526+1G>A showed the activation of a cryptic donor site, causing loss of the last 31 nucleotides of exon 2. In mutant c.714-1G>A, the major splicing event showed loss of 38 nucleotides in exon 4, due to activation of a novel cryptic acceptor site. In the event of c.956-2A>G, sequencing showed a complete skipping of the subsequent exon 5. The c.1502-6G>A variant causes activation of a cryptic acceptor site in intron 7 which leads to a four-nucleotide retention of the intronic sequence. The consequences of all variants were additionally evaluated using in silico splice site predictions by the integrated software tool Alamut v.2.6. Predictions of cryptic sites and the in vitro nucleotide changes observed were fully in agreement. These four intronic HNF1A variants likely cause MODY3 due to abnormally spliced mRNAs. By combining in silico predictions and the pCAS2 minigene expression system, we here provide further evidence of the variants role as pathogenic MODY3 mutations.

Disclosure

M.H. Solheim: None. M. Nasrollahzadeh Khakiani: None. J. Molnes: None. I. Aukrust: None. P. Njølstad: None. S. Johansson: None.
2470-PUB: Standardized Glucose, Insulin, and ß-Cell Response to Glucose Reveal Important Details of the Pre-DM Trajectories Not Shown in Retrospective or Prospective Epidemiological Analyses
Authors: HANSEN BARBARA C. , NEWCOMB JENNIFER D. , LIN PAUL K. , CATHERINE JEN K-L.
Published: June 2019

Over the past 30 years, in prospective longitudinal studies of the development of T2DM and its risk factors, within-subject individual variable trajectories (as characterized in nonhuman primates (NHPs) have shown very different individual profiles compared to data synchronized retrospectively on the time point of DM diagnosis (DM DX) (e.g., Whitehall II or ARIC studies). About 30% of unrelated NHPs naturally and spontaneously develop T2DM at an average age of 19.2 ± 5.4 years (equivalent to ∼60 years in humans). New trajectory analyses of the standardized fasting glucose (FPG), fasting insulin (FPI) and β- cell function of those monkeys that became DM while under longitudinal study indicate that the functional β-cell changes in the pre-DM period, applying the usual epidemiological analytical approach used in humans, is likely to be highly misleading. The NHP DM progression data of 24 adult monkeys studied for an average of 8 years, with pre-DM data obtained up to 18 years pre-DM DX show that these trajectories must be analyzed individually and then standardized for rate of progression, timing of progression and amplitude of changes in order to establish the valid naturally-occurring patterns of the pre-DM progression seen below.

Disclosure

B.C. Hansen: None. J.D. Newcomb: None. P.K. Lin: None. K. Jen: None.

Funding

National Institutes of Health
2471-PUB: Evaluation of Model-Based Indices of Glucose Metabolism in Morbidly Obese Subjects
Authors: BRIX JOHANNA , KRZIZEK EVA-CHRISTINA , TURA ANDREA , PACINI GIOVANNI , LUDVIK BERNHARD
Published: June 2019

Patients with MO are insulin resistant and the degree of insulin resistance (IR) further depends on glycemic status. To determine IR, model-based indices use fasting and/or post-challenge glucose and insulin levels. In this study, we examined the validity of indices of GM in a large cohort of patients with MO and varying degrees of glucose tolerance. We included 1337 patients with MO without known diabetes (mean age 40±12 years, mean±SD, BMI 45.0± 6.6kg/m²). All patients underwent a 75g oGTT, glucose and insulin levels were repeatedly measured for the calculation of IR and Insulin Sensitivity indices (Table) and analysis for the respective glucose tolerance categories. Diabetes mellitus (DM) was detected in 120 (9%) patients, 348 (26%) had impaired glucose tolerance or impaired fasting glucose (IGT/IFG), 869 (65%) patients had normal glucose tolerance (NGT). Patients with DM were older (p<0.001) and had a lower BMI (p= 0.014) compared to NGT. GM indices are depicted in the Table. and show that all indices except for Matsuda and AUC insulin show differences between the glucose tolerance categories. In patients with MO, GM indices using basal glucose and insulin levels can be used to routinely measure insulin resistance without need of more elaborate testing. In particular, they can be used in patients with diabetes when an OGTT would be inappropriate.

Disclosure

J. Brix: None. E. Krzizek: None. A. Tura: None. G. Pacini: None. B. Ludvik: Advisory Panel; Self; Amgen Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Sanofi, Takeda Development Centre Europe Ltd. Research Support; Self; Akebia Therapeutics, Eli Lilly and Company, Novartis AG, Novo Nordisk A/S. Speaker's Bureau; Self; Merck Sharp & Dohme Corp.
2473-PUB: Differential Effects of High-Fat Diet and Niacin on Nonalcoholic Fatty Liver Disease in C57BL/6J and B6129SF2/J Mice: A Metabolomic Analysis
Authors: FANG HAN , LI ZHUOYUE , GRAFF EMILY , MCCAFFERTY KAYLEEN J. , JUDD ROBERT L.
Published: June 2019

Recent studies have demonstrated significant metabolic changes in different strains of mice fed a high-fat diet (HFD). Pharmacological doses of niacin can improve nonalcoholic fatty liver disease (NAFLD) in HFD-fed rodents. However, previous study from our lab demonstrated that niacin increases the hepatic triglyceride content and histological score in HFD-fed B6129SF2/J (B6129) mice but has no effect in C57BL/6J (B6) mice. Therefore, the aim of this study was to identify the mechanism responsible for niacin-induced fatty liver in B6129 mice using targeted liver metabolomic analysis. Livers were collected from B6 and B6129 mice, which were fed either a chow (10% fat) or HFD (60% fat) for 20 weeks with niacin (360 mg/kg/day) or vehicle supplementation from week 5 to week 20. Two hundred polar metabolites were identified in these livers. A total of 21 and 26 metabolites identified as different between chow and HFD group in B6 and B6129 mice, respectively. In B6 mice, only 6 metabolites were identified as different with niacin treatment and no specific pathways appeared to be impacted. In contrast to B6 mice, 16 metabolites and 5 pathways were impacted by niacin in HFD-fed B6129 mice. Of the metabolites that were changed in these pathways, only hydroxyphenylpyruvate (also known as 4-Hydroxyphenylpyruvate, 4-HPP) did not change with HFD feeding but significantly decreased with niacin treatment. 4-HPP is an intermediate in the metabolism of phenylalanine/tyrosine and improves mitochondrial oxidation in rats subjected to hemorrhagic shock. Therefore, decreased 4-HPP by niacin may lead to decreased beta-oxidation in the liver and subsequent lipid accumulation and liver damage in B6129 mice. Further lipidomic analysis suggested different hepatic lipid profiles with HFD and niacin treatment in B6129 mice, which may explain the increased NAFLD development in these mice with niacin treatment.

Disclosure

H. Fang: None. Z. Li: None. E. Graff: Research Support; Self; Elanco Animal Health. K.J. McCafferty: None. R.L. Judd: None.

Funding

Boshell Diabetes and Metabolic Diseases Research Program
249-LB: Identification of Hybrid Insulin Peptides through Mass Spectrometric Analyses
Authors: DELONG THOMAS
Published: June 2019

Hybrid insulin peptides (HIPs) form if peptide fragments of (pro)insulin bind through peptide bonds to fragments of other proteins. We previously demonstrated that HIPs can be detected in human and murine islets by mass spectrometry, are targeted by diabetes-triggering CD4 T cell clones from non-obese diabetic mice, and are recognized by CD4 T cells isolated from the residual islets of pancreatic organ donors with type 1 diabetes (T1D). HIPs may be key antigens in the development of T1D as they contain non-genomically encoded amino acid sequences. Since the initial discovery of HIPs, we have established a set of rigorous criteria that allow us to identify various new HIPs in pancreatic islets with high confidence. The formation of HIPs involves the chemical activation of the C-terminal carboxylic acid groups of (pro)insulin peptides. These activated peptides can react with N-terminal amines of other peptides, leading to the formation of HIPs. The identification of such activated peptides is of great interest as it will help us understand which peptides contribute to hybrid peptide formation. We developed a new technique, which allows us to identify such chemically activated peptides by mass spectrometry. This technique may lead to the identification of activated peptides that are not derived from (pro)insulin and could provide valuable information needed for the identification of new hybrid peptides that may play a role in the development of type 1 diabetes and other autoimmune diseases.

Disclosure

T. Delong: None.

Funding

American Diabetes Association (1-15-ACE-14); National Institutes of Health; JDRF
2491-PUB: Proinsulin C-Peptide Attenuates Vascular Dysfunctions by Activating Nrf2
Authors: LAN FAN , LIN YAN , GAO ZHENFENG , ZHAO PAN , ZHANG XINGYU , SHIRAISHI YASUNAGA , IDO YASUO
Published: June 2019

We and others demonstrated that insulin C-peptide has biological activities and may prevent tissue damages by diabetes. Our research using STZ diabetic rats and other models suggested that diabetic vascular dysfunctions, e.g., increased vascular permeability and blood flows are caused by oxidative and nitrosative stress initiated from increased NADH redox (reductive stress). However, C-peptide prevented dysfunctions without normalizing redox state and worked in nondiabetic rats. In this study, we further examined modes of actions and investigated its mechanisms. Male Wistar rats were used in animal study. Human de-differentiated adipocytes (DFAT cells) were used for cell culture study. Lentivirus vector expressing LC3-GFP was created to assess autophagy. Regional blood flow was assessed using microsphere. Acute hyperglycemia induced by glucose infusion for 5 hours resulted in increased regional blood flows in retina, sciatic nerve and kidney. Simultaneous infusion of C-peptide up to 16 mg/kg did not prevent the increases, while twice daily subcutaneous injection of 400 µg/kg for 2-days prior to infusion prevented completely. One-day injection was only slightly effective. Bolus i.v. injection of L-lactate 1 mol/kg but not NaCl caused reductive stress and blood flow increase, and this was also prevented by twice daily 2-days injection of C-peptide. Thus, C-peptide appeared to induce some factors counteracting oxidative stress during 2 days. In DFAT cells, LC3-GFP puncta was increased 3-fold by one time 100 nM C-peptide treatment in 1 hour. This was followed by nuclear localization of Nrf2 transcription factor at 2 hours. At 4 hour, 4-fold increase in Nrf2 target genes, IDH and SRXN1, expression were observed (p<0.05). Similar 35-62% increase in Nrf2 target genes (Idh, Srxn1, Pgd and Hmox) were observed in rat aorta after twice daily 2-days injection of C-peptide. These results suggest that injections of pharmacological dose of C-peptide increase Nrf2 antioxidants expression through activating autophagy.

Disclosure

F. Lan: None. Y. Lin: None. Z. Gao: None. P. Zhao: None. X. Zhang: None. Y. Shiraishi: None. Y. Ido: None.

Funding

Japan Society for the Promotion of Science
2493-PUB: Distinct 5-Methylcytosine Profiles in Ribo-Minus RNA from Mouse Brown Adipose Tissue
Authors: ZHANG CHUANHAI , YIN RUILI , WANG JINGJING , QUANWEI WEI
Published: June 2019

Methylation of DNA and RNA has become a research hotspot because of its diverse regulation of biological function and processes. In particular, posttranscriptional modifications in RNA, including methylation of the N6 positions in adenine (m6A), N1 positions in adenine (m1A) and methylation of carbon 5 in cytosine (m5C). However, information about the transcriptome-wide distribution and possible functions of m5C in different cell types, tissues, and organisms is still extremely limited. Brown adipose tissue is a functional organ for thermogenesis and an important potential target for the treatment of obesity and diabetes. The insights into possible functions of m5C modification and implications in BAT are significant and urgent to be revealed. In our study, we performed an unbiased global analysis of m5C in total and Ribo-minus RNA of brown adipose tissue from mouse after thermoneutrality (29?-30?) and/or cold stimulation (4?) for 1 day. We identified total 8153 sites in mRNA, 570 sites in CircRNA, and 991 sites in LncRNA, with respect to the degree of methylation, functional classification of methylated transcripts, and position bias within the transcript. Interestingly, there are intriguing differences in BAT after thermoneutrality and/or cold stimulation, which may provide us with new ideas to activate or enhance functionality of BAT. Our findings show the first comprehensive picture of m5C in the BAT from the mouse. These data establish and provide an important resource for future studies of function and biological significance of m5C in Ribo-minus RNA in brown adipose tissue.

Disclosure

C. Zhang: None. R. Yin: None. J. Wang: None. W. Quanwei: None.

Funding

National Natural Science Foundation of China (81700684 to C.Z.), (31402075), (31572403)
250-OR: Beta-Cell Rest Induced by Dapagliflozin Improves ß-Cell Secretory Capacity in Type 2 Diabetes Patients: A Randomized Controlled Trial vs. Gliclazide
Authors: VAN BOMMEL ERIK J.M. , MUSKIET MARCEL H. , VAN BAAR MICHAEL JACOBUS , KRAMER MARK H. , NIEUWDORP MAX , VERCHERE CAMERON B. , VAN RAALTE DANIËL H.
Published: June 2019

Introduction: Disease progression in type 2 diabetes (T2D) is due to a continuous loss of β-cell function, partly driven by chronic secretory stress on remaining islets caused by hyperglycemia. Beta-cell loss may be further enhanced by insulin secretagogues such as sulfonylureas. In contrast, sodium-glucose cotransporter-2 inhibitors (SGLT2is) may improve beta-cell function by reducing β-cell afterload by lowering blood glucose levels through glucosuria.

Methods: Forty-four T2D patients treated with metformin (age 63 ± 7 years; HbA1c 7.3 ± 0.6%; GFR 113±19 mL/min) were randomized to dapagliflozin 10mg (DAPA, n=24) or gliclazide 30mg (GLIC, n=20) QD. At baseline and week 12, whole-body insulin sensitivity (M-value) and β-cell function were quantified by gold-standard hyperinsulinemic-euglycemic and hyperglycemic clamp with additional arginine stimulation. First-phase glucose-stimulated and arginine-stimulated incremental area under the C-peptide curves (iAUCCP) were calculated. iAUCCP multiplied by M-value rendered disposition indices (1st phase and ARG-DI; nmol.min/L.mg/kg.min).

Results: HbA1C decreased similarly (-0.47% with DAPA and -0.65% with GLIC; p=ns). DAPA, but not GLIC, decreased fasting hyperinsulinemia (-18.4 pmol/l vs. +2.7 pmol/l, p=0.01). Neither treatment improved insulin sensitivity. GLIC significantly increased 1st phase DI from 0.75 ± 1.21 to 2.89 ± 3.25 (p<0.01), while DAPA did not (1.51 ± 2.81 to 2.15 ± 2.27, p=0.29). ARG-DI increased from 42.4 ± 12.1 to 45.2 ± 15.6 (p=0.03) with DAPA but was not improved by GLIC (52.3 ± 9.9 to 63.7 ± 8.6; p=0.28).

Conclusion: In contrast to the secretagogue GLIC, DAPA lowered fasting insulin levels and did not further enhance C-peptide secretion in response to hyperglycemia. However, it did improve ARG-DI, which may reflect β-cell total secretory capacity. Thus, by lowering beta-cell afterload, DAPA may confer long-term β-cell benefit versus GLIC.

Disclosure

E.J.M. van Bommel: None. M.H. Muskiet: Consultant; Self; Eli Lilly and Company, Novo Nordisk A/S, Sanofi. M.J. van Baar: None. M.H. Kramer: None. M. Nieuwdorp: Advisory Panel; Self; Caelus health. C.B. Verchere: Advisory Panel; Self; Sirona Biochem. Board Member; Self; Integrated Nanotherapeutics. D.H. van Raalte: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Merck Sharp & Dohme Corp., Sanofi. Research Support; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Merck Sharp & Dohme Corp., Sanofi.

Funding

AstraZeneca Nederland BV
251-OR: Relationship of Short-Term HbA1c Change with Vascular Events, Severe Hypoglycaemia, and Mortality
Authors: NANAYAKKARA NATALIE , OHKUMA TOSHIAKI , WOODWARD MARK , CHALMERS JOHN , ZOUNGAS SOPHIA
Published: June 2019

Objective: To examine the effects of change in glycaemic control on clinical outcomes in patients with type 2 diabetes.

Research Design and Methods: Data were obtained from 9981 participants of the ADVANCE trial and ADVANCE-ON follow-up. Participants were categorized into 3 groups by glycated haemoglobin (HbA1c) change from baseline to 6 months, >0.25% decrease, >0.25% increase and minimal change (±0.25%). Adjusted Cox and logistic regression analyses examined relationships between HbA1c change and all-cause mortality, macrovascular events, microvascular events and severe hypoglycaemia.

Results: Mean (±SD) age was 65.8±6.4 years, diabetes duration 7.9±6.4 years and HbA1c 7.5±1.6% at baseline. In the first 6 months, 48%, 22% and 30% of participants had an HbA1c decrease, minimal change or increase respectively. Median (IQR) follow-up was 9.4 (5.1-10.4) years. Compared to those with minimal HbA1c change at 6 months, participants with an HbA1c increase at 6 months had greater risk of risk of all-cause mortality [1.26 (1.11-1.43)], macrovascular events [1.26 (1.10-1.44)] and microvascular disease [1.36 (1.09-1.70) whereas participants with an HbA1c decrease did not have greater risk of all-cause mortality [0.99 (0.87-1.12)], macrovascular events [1.06 (0.93-1.21)] or microvascular disease [0.97 (0.77-1.21)]. Neither HbA1c increase [1.05 (0.83-1.33)] or decrease [1.16 (0.91-1.47)] was associated with greater risk of severe hypoglycaemia. These effects were similar when HbA1c change at 3 months was considered and in subgroup analyses by age, gender, diabetes duration, or baseline HbA1c.

Conclusions: Increased rather than decreased HbA1c at 6 months was associated with increased risks of death and vascular events in type 2 diabetes.

Disclosure

N. Nanayakkara: None. T. Ohkuma: None. M. Woodward: Consultant; Self; Amgen Inc., Kirin. J. Chalmers: Research Support; Self; Idorsia. S. Zoungas: None.
259-OR: Magnetic Resonance Imaging of Transplanted Islets Labeled with Exendin-4-Conjugated Manganese Magnetism-Engineered Iron Oxide Nanoparticles
Authors: JUANG JYUHN-HUARNG , SHEN CHIA-RUI , WANG JIUN-JIE , WU SHU-TING , CHEN CHEN-YI , KAO CHEN-WEI , LIN SUNG-HAN , WANG YUN-MING
Published: June 2019

To detect and trace transplanted β-cells by magnetic resonance imaging (MRI), we conjugated manganese magnetism-engineered iron oxide (MnMEIO) nanoparticles with exendin-4 (MnMEIO-Ex4) which can specifically bind glucagon-like peptide-1 receptors on β-cells. Incubation of MIN6 β-cells and MnMEIO showed MnMEIO with iron content 5-40 μg/mL had no adverse effect on MIN6 β-cells assessed by the fluorescence of propidium iodide and insulin secretory response to glucose. Positive iron staining by Prussian blue was found in the MIN6 loaded with MnMEIO-Ex4 but not with MnMEIO. A transmission electron microscope with elementary mapping of iron and manganese demonstrated MnMEIO-Ex4 nanoparticles distributed in the cytoplasm of MIN6 β-cells. In vitro 7.0 T MR image showed loss of intensity in MIN6 β-cells loaded with MnMEIO-Ex4 but not with MnMEIO. With addition of poly-L-lysine, only MIN6 β-cells incubated with MnMEIO-Ex4 but not MnMEIO stimulated proinsulin mRNA expression and insulin secretion. On day 1, 6, 15, 22 and 29 after syngeneic transplantation of 700 C57/B6 mouse islets labeled with MnMEIO-Ex4, the islet graft under left kidney capsule could be visualized on 7.0 T MRI as persistent hypodense areas (indicated by arrows in Figure). Our results indicate MnMEIO-Ex4 nanoparticles are safe and effective for the detection and monitoring of islet grafts by MRI.

Disclosure

J. Juang: None. C. Shen: None. J. Wang: None. S. Wu: None. C. Chen: None. C. Kao: None. S. Lin: None. Y. Wang: None.

Funding

Chang Gung Memorial Hospital
260-LB: Continuous Glucose Monitoring Metrics in Islet Transplant Recipients with Long-Term Insulin Independence
Authors: BAIDAL DAVID , ALVAREZ GIL ANA M. , PADILLA NATHALIA , RICORDI CAMILLO , ALEJANDRO RODOLFO
Published: June 2019

Continuous glucose monitoring (CGM) following islet transplantation (ITx) has shown improved time-in-range, reduced glycemic variability (GV) and hypoglycemia prevention despite subnormal beta-cell function. However, it is unclear if improvement by CGM persists long-term in insulin independent (Ifree) ITx recipients. We evaluated 5 ITx recipients who remained Ifree for >5 years. Subjects completed 7-day non-blinded CGM (Dexcom G4 Platinum). HbA1c, 90min C-peptide, Clarke score, and BETA-2 score (composite measure of beta-cell function after ITx) were obtained. Before ITx, mean age was 47.2±4.0 years, diabetes duration 34.6±9.4 years, HbA1c 7.0±1.2%, insulin/Kg/day 0.44±0.08 units and Clarke score 5 (range 4-7). Subjects received 1-2 islet infusions. Induction immunosuppression consisted of ATG or daclizumab and dual maintenance with sirolimus, tacrolimus or mycophenolate mofetil. At time of CGM, subjects were Ifree and 10.7±3.5 years post-ITx (range 7.7-16.0). Two subjects were on non-insulin adjunct therapies. CGM and metabolic data, BETA-2 and Clarke scores are shown in Table 1. In Ifree ITx recipients with very good long-term graft function (BETA-2 score>15) and restoration of hypoglycemia awareness, CGM shows near-normalization of time-in-range and GV with minimal time-in-hypoglycemia. ITx is a successful long-term cellular therapy for T1D in a selected group of patients.

Disclosure

D. Baidal: None. A.M. Alvarez Gil: None. N. Padilla: None. C. Ricordi: Advisory Panel; Self; Zone Labs. R. Alejandro: None.

Funding

National Institute of Allergy and Infectious Diseases/National Institute of Diabetes and Digestive and Kidney Diseases (U01-DK-070460); Immune Tolerance Network (NIS01); Diabetes Research Institute Foundation; State of Florida; University of Miami Clinical and Translational Science Institute (1UL1TR000460); JDRF International (4-2004-361)
268-OR: Insulin Resistance in the Liver, but Not Muscle or Adipose, Is Essential for Remission from Newly Diagnosed Type 2 Diabetes by Short-Term Insulin Intensive Therapy
Authors: WAN XUESI , HUANG ZHIMIN , LI YANBING
Published: June 2019

To clarify the effects of insulin resistance from different tissues on the remission from newly diagnosed type 2 diabetes (T2DM) following short-term insulin intensive therapy (SIIT). This study enrolled 37 healthy controls and 160 patients with newly diagnosed type 2 diabetes mellitus (n=160, HbA1c11±2.1%). They were randomly divided to four groups, CSII alone for 2 weeks, or combination with an insulin sensitizer (CSII + metformin or CSII + rosiglitazone), or an antioxidant (CSII + α-lipoic acid). HOMA-IR, Adipo-IR and intramyocellular lipid (IMCL), were compared before and after termination of CSII. After short-term intensive treatment, 77.5% patients achieved normal HOMA-IR but little change in the Adipo-IR. The IMCL were nearly normalized except for those in metformin group, impacted by the lower insulin dose. The improvement of HOMA-IR and FFA, not IMCL or Adipo-IR, statistically related to the percentage declines of FPG (r=0.317 and 0.275), while only the HOMR-IR was associated with ΔPPG% (r=0.284). And 40.6% patients achieved sustained normal HOMA-IR in the first 3 months after CSII and attained 50% remission rate at the 5-year, far beyond 8.9% in the other patients (HR = 2.82 95% CI is1.813-4.388, p<0.001). Besides, slight superiority has occurred with HOMA-IR, Adipo-IR and IMCL in the CSII+ rosiglitazone group. In brief, short-term CSII therapy, with or without insulin sensitizers, is extremely effective in improving insulin resistance, even normalizing the HOMA-IR and IMCL. Sustained normal hepatic insulin resistance is essential for remission from T2DM. Insulin reversed ectopic fat in muscle with dose effect and the combination therapy with rosiglitazone is superior.

Disclosure

X. Wan: None. Z. Huang: None. Y. Li: None.
276-LB: Constitutive Activation of PKA Signaling in POMC-Expressing Cells Improves Systemic Glucose Metabolism
Authors: CHOI JONG HAN , MIN SE HEE , KANG GIL MYOUNG , KIM JI YE , KIM MIN-SEON
Published: June 2019

Constitutive Proopiomelanocortin (POMC)-expressing cells are mainly found in the hypothalamic arcuate nucleus, brainstem solitary tract nucleus, and pituitary gland. The cAMP-dependent protein kinase A (PKA) critically transduces signaling pathways of many important hormones and neurotransmitters including glucagon, GLP-1, α-MSH, dopamine and catecholamine. We generated the mice deleting PKA regulatory subunit R1A (Prkar1a) in POMC-expressing cells to induce constitutive activation of PKA signaling in POMC cells. These mice exhibited interesting phenotypes similar to human Cushing's syndrome; thin skin, hair loss, increased fat mass but reduced muscle and bone mass, female infertility, retarded linear growth, hump and insulin resistance. Moreover, they had increased corticosterone levels both in urine and plasma along with adrenal cortical hypertrophy. Interestingly, despite severe insulin resistance and obesity, plasma glucose concentrations were significantly lower in both fasting and post-glucose loading conditions. These mice showed enhanced insulin-independent glucose disposal. FDG-PET scanning and 2DG uptake tests revealed increased glucose uptake in the brain and the kidney. Normalization of corticosteroid levels by bilateral adrenalectomy and corticosteroid replacement could not normalize blood glucose. Collectively, our hypothesis is that abnormal activation of PKA signaling in pituitary POMC cells causes human Cushing’s syndrome-like phenotype whereas unusual activation of PKA signaling in hypothalamic POMC neurons enhances glucose disposal in the brain and the kidney.

Disclosure

J. Choi: None. S. Min: None. G. Kang: None. J. Kim: None. M. Kim: None.

Funding

National Research Foundation of Korea
276-OR: Rethinking Metabolically Healthy Obesity: What Is the Underlying Risk?
Authors: AXELROD CHRISTOPHER L. , HARI ADITHYA , MEY JACOB T. , BEYL ROBBIE A. , KIRWAN JOHN P.
Published: June 2019

Introduction: Type 2 diabetes (T2D) is a multifactorial disease with both heritable and environmental origins. Overweight and obesity are the primary risk factors, present in 90% of persons with T2D. However, little is known of the intrinsic risk carried by individuals with moderate to severe obesity. The purpose of this study was to identify and analyze risk factors in obese individuals with and without clinical metabolic risk factors. We hypothesized that the insulin sensitivity of metabolically healthy obese individuals would be similar to their BMI matched counterparts.

Methods: Eighty-seven sedentary adults underwent an inpatient stay consisting of anthropometric measurements, blood chemistries, body composition (DXA), aerobic capacity (VO2MAX), and insulin sensitivity testing (glucose clamp). Subjects were first stratified by BMI (<25 and >30). Those with a BMI >30 were further stratified based upon a composite metabolic health score consisting of: BMI, VO2MAX, HOMA-IR, triglycerides, and cholesterol levels. Group differences were assessed using a one-way ANOVA with multiple comparisons.

Results: Individuals with obesity had elevated HDL, LDL, blood pressure, waist-to-hip ratio and body fat (ALL P<0.05) compared to lean controls, with no differences between metabolically healthy and unhealthy individuals. Metabolically unhealthy individuals had elevated fasting insulin (P<0.05) and glucose (P<0.05) levels compared to both lean controls and metabolically healthy obese individuals. However, both the metabolically healthy and unhealthy obese individuals had reduced insulin sensitivity (lean to obese P<0.01, interaction P=0.40) compared to lean controls.

Conclusions: Metabolically healthy obese individuals retain the risk associated with T2D, most notable being reduced insulin sensitivity. Furthermore, we posit that safe and effective treatments for T2D, such as insulin sensitizing therapy or bariatric surgery, may be warranted in metabolically healthy obese individuals alike.

Disclosure

C.L. Axelrod: None. A. Hari: None. J.T. Mey: None. R.A. Beyl: None. J.P. Kirwan: None.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases; National Institute on Aging; Louisiana Clinical & Translational Science Center
277-LB: LRP1 Mediates the Transport of Leptin Across the Blood-CSF Barrier at the Choroid Plexus
Authors: SEO JI A , KANG MIN-CHEOL , MENDES NATALIA FERREIRA , ARAUJO THIAGO , YANG WON-MO , KIM YOUNG-BUM
Published: June 2019

Leptin is an adipocyte-derived hormone that reduces food intake and stimulates energy expenditure. In order to achieve its physiological actions, it needs to enter the brain. The precise routes of brain entry of many metabolic signals have not been identified, but most studies suggest that transport across the BBB and CSF barrier are required. This study was designed to test the hypothesis that LRP1 mediates leptin’s transport across the blood-CSF barrier at the choroid plexus (ChP). To address this, we have first generated Foxj1-Cre; tdTomato mice to see the expression pattern of Foxj1 in the brain. The expression of tdTomato is restricted to the epithelial cells of the ChP and the ependymal cells of the ventricles. Mice lacking LRP1 in the epithelial cells and ependymal cells of the brain were thus generated and studied. Here we show that deletion of LRP1 in the epithelial cells of the ChP and the ependymal cells of the ventricles causes hyperleptinemia. This effect is independent of body weight and adiposity. In addition, mice lacking LRP1 in the epithelial cells of the ChP and the ependymal cells of the ventricles are insulin-resistant. Most importantly, leptin-induced food intake and hypothalamic Stat3 phosphorylation were significantly impaired in Foxj1-Cre; LRP1lox/lox mice compared with LRP1lox/lox mice. However, ICV leptin decreased food intake and increased hypothalamic Stat3 phosphorylation in Foxj1-Cre; LRP1lox/lox mice. There were no differences in both food intake and Stat3 phosphorylation between LRP1lox/lox mice and Foxj1-Cre; LRP1lox/lox mice. These data clearly demonstrate that LRP1 in the epithelial cells of the ChP mediates leptin transport across the blood-CSF barrier at the ChP and further implicate that defective leptin transport may not contribute to the regulation of body weight homeostasis.

Disclosure

J. Seo: None. M. Kang: None. N. Ferreira Mendes: None. T. Araujo: None. W. Yang: None. Y. Kim: None.

Funding

National Institutes of Health (R01DK106076); National Research Foundation of Korea (2018R1D1A1B07049123)
279-OR: How to Use rtCGM Data to Predict Future Severe Hypoglycemia?
Authors: HERMANNS NORBERT , EHRMANN DOMINIC , CALHOUN PETER , FRECKMANN GUIDO , HEINEMANN LUTZ
Published: June 2019

We combined rtCGM-baseline-data of the HypoDE and DIAMOND study to predict future severe hypoglycemia (SH; third party assistance for recovery). At baseline, in both studies, the rtCGM system (Dexcom G4) was masked. We analyzed the data of 127 study participants in the control groups with type 1 diabetes (age 46.0 ±11.5 years; diabetes duration 22.4 ±14.1 years, A1c 7.9 ±1.0%) using SMBG during the trials. According to the consensus statement we used the following hypoglycemia-specific predictors: % of glucose values < 70mg/dl and <54 mg/dl, the number of such events, Low Blood Glucose Index (LBGI), and the coefficient of variation (CV). After baseline, a total of 44 severe hypoglycemic events were observed during the following 6-months; with 12.6% affected participants. We analyzed the area under the Receiver Operating Characteristics (ROC) curves and determined cut-off values with an optimal sensitivity and specificity (Table). All hypoglycemia-related parameters, except CV, were able to predict SH during the next 6 months significantly better than prediction by chance; e.g., if ≥ 7% of glucose values were < 70 mg/dl, the probability for SH in the next 6-months was 23.4%. CGM-derived hypoglycemic outcome parameters might help to identify people with diabetes who have an increased risk for future hypoglycemia. These results should be corroborated by non-masked use of rtCGM but might also be useful in patients using SMBG.

Disclosure

N. Hermanns: Advisory Panel; Self; Abbott. Board Member; Self; Eli Lilly and Company. Research Support; Self; Abbott, Berlin-Chemie AG, Dexcom, Inc., Roche Diabetes Care. Speaker's Bureau; Self; Berlin-Chemie AG, Dexcom, Inc., Novo Nordisk A/S. D. Ehrmann: Advisory Panel; Self; Medtronic. Speaker's Bureau; Self; Abbott, Berlin-Chemie AG, Dexcom, Inc. P. Calhoun: Stock/Shareholder; Self; Dexcom, Inc. G. Freckmann: Advisory Panel; Self; Abbott, Novo Nordisk Pharma GmbH, Sensile Medical AG. Consultant; Self; Metronom Health Europe, Roche Diabetes Care, Sanofi. Speaker's Bureau; Self; Ascensia Diabetes Care, Dexcom, Inc., Ypsomed AG. L. Heinemann: Consultant; Self; Roche Diabetes Care. Research Support; Self; Dexcom, Inc. Stock/Shareholder; Self; Profil Institute for Metabolic Research, ProSciento.

Funding

Dexcom, Inc.
285-LB: Short-Term Effect of Saturated and Monounsaturated Fatty Acids on Hepatic Energy Metabolism
Authors: BEDNARSKI TOMASZ , RAHIM MOHSIN , YOUNG JAMEY
Published: June 2019

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease affecting about 30% of the U.S. population. The prognosis for simple liver steatosis is relatively benign; however, chronic inflammation and progressive fibrosis may lead to nonalcoholic steatohepatitis (NASH) and later to cirrhosis and hepatocellular carcinoma. There is no established treatment to stop progression from NAFLD to NASH to date. Therefore, we developed a protocol to investigate the acute impact of saturated (SFA) and monounsaturated (MUFA) fatty acids on liver energy metabolism and their potential lipotoxic effects. Conscious, unrestrained, overnight fasted 12 weeks old male C57Bl/6J mice were given continuous, intravenous infusions of either saline, 20% lard oil or 20% olive oil emulsions (as described in Stein DT et al. J Clin Invest. 1997; 100 (2): 398-403) for 5 hrs. Lipid infusion was accompanied by primed, continuous infusion of [6,6-2H2] glucose (16mg/kg/min for 5 min and 0.8 mg/kg/min continuously) and sodium [13C3]lactate (200 μM/kg/min 5 min and 40 μM/kg/min continuously) intravenously for 4 and 2 hrs, respectively. Both plasma and liver free fatty acids were upregulated in SFA and MUFA groups. However, liver triglycerides, alanine aminotransferase (ALT) and aspartate transaminase (AST) were significantly increased, whereas plasma insulin and glucose levels significantly decreased, only in the MUFA group. 13C and 2H-enrichment of plasma glucose and liver alanine, glutamate, lactate and urea obtained at the end of the study were measured by GC-MS and used to regress liver metabolic fluxes using a flexible flux modeling platform (INCA). We observed that both SFA and MUFA increased the absolute rate of dihydroxyacetone phosphate (DHAP) flux from glycerol. Absolute flux of acetyl-CoA production was shifted from synthesis through pyruvate dehydrogenase toward increased contribution from fatty acid oxidation. On the other hand, only MUFA decreased the gluconeogenesis rate.

Disclosure

T. Bednarski: None. M. Rahim: None. J. Young: Consultant; Self; Pfizer Inc. Stock/Shareholder; Self; Metalytics.

Funding

National Institutes of Health (R01DK106348)
287-LB: Autophagic Degradation of CRY1 and Control of Gluconeogenesis
Authors: SOLER MIRIAM TOLEDO , SINGH RAJAT
Published: June 2019

Cryptochrome 1 (CRY1) is a core clock repressor that maintains clock periodicity. CRY1 robustly suppresses hepatic gluconeogenesis by regulating CREB/cAMP and glucocorticoid receptor signaling and by reducing levels of FoxO1—a transcription factor that drives gluconeogenesis. Autophagy degrades cytoplasmic contents in lysosomes for maintenance of quality control and provision of energy. Since the liver clock and autophagy are each regulated by insulin and glucagon, we hypothesized that the control of autophagy, the circadian clock, and glucose homeostasis are interlinked. We have shown that induction of circadian autophagy between 3pm-7pm degrades CRY1, which releases its inhibition of gluconeogenesis and promotes glucose production. Acutely blocking autophagy or inactivating the LC3-Interacting-Region (LIR) motifs of CRY1, which allow it to interact with the autophagy marker LC3, led to accumulation of CRY1 and suppression of gluconeogenesis. Thus, autophagy drives glucose production in liver by timed degradation of CRY1. Interestingly, high-fat feeding accelerates the autophagic degradation of CRY1, which contributes to obesity-associated hyperglycemia. Since phosphorylation of CRY1 triggers its proteasomal degradation, and since phosphorylation in the vicinity of LIRs is thought to facilitate interaction with LC3, we hypothesized that phosphorylation of CRY1 leads to its degradation through autophagy and increases glucose production. Accordingly, we generated specific phosphorylation-resistant and constitutively-phosphorylated CRY1 mutants that were delivered to the liver. While the phosphorylation-resistant CRY1 mutant accumulated in liver and lowered blood glucose levels, the constitutively-phosphorylated CRY1 mutation that favors its rapid degradation resulted in increased blood glucose. Our results show that CRY1 phosphorylation couples its turnover by autophagy to regulation of gluconeogenesis—revealing novel targets to prevent obesity-associated hyperglycemia.

Disclosure

M. Toledo Soler: None. R. Singh: None.

Funding

American Diabetes Association (1-18-IBS-062 to R.S.)
287-OR: Acute Exercise Enhances the Differentiation Rate of Human Adipocyte Precursor Cells
Authors: VARSHNEY PALLAVI , GUTH LISA M. , ANDERSON CARA M. , RAAJENDIRAN ARTHE , WATT MATTHEW J. , HOROWITZ JEFFREY F.
Published: June 2019

The direct effects of exercise on white adipose tissue morphology and metabolic function remain unclear, due in large part to confounding effects of the reduction in fat mass that often accompanies exercise training. The purpose of this study was to determine if a session of endurance exercise could impact the rate of adipogenesis. Eight lean, healthy human subjects performed a single session of exercise (1 hour of endurance exercise at 65% of their maximal aerobic capacity; VO2max). Blood samples were collected without prior exercise (No-EX) and immediately after exercise (EX). Serum was extracted, and human adipocyte precursor cells were incubated for 72 hours in vitro in either the No-EX or EX serum. This serum preincubation was immediately followed by a standard 14-day differentiation protocol. On both days 3 and 9 of differentiation, mRNA expression levels of differentiation markers, PPARG and FABP4, were significantly greater (P<0.01) in cells preincubated in the EX vs. No-EX serum (e.g., Day 3 PPARG expression: 11.4±0.6 vs. 8.3±0.4 2-∆Ct; P=0.005, and Day 9 FABP4 expression: 2185±82 vs. 1619±98 2-∆Ct; P<0.0001). In addition, after 14 days of differentiation, we also found significantly greater protein abundance of important lipolytic and esterification regulators, G0S2, ATGL, HSL and GPAT1 (all P<0.05). As expected, compared with No-EX, the EX sera had greater concentrations of IL-6, lactate, fatty acids, epinephrine, and growth hormone, some of which may contribute to the enhanced adipogenic signaling after exercise.

In summary, factors released into the circulation during and/or immediately after a session of endurance exercise can accelerate the rate of differentiation in human adipocyte precursor cells. Enhanced adipogenic signaling in response to exercise may have metabolic health benefits by priming adipose tissue for a more effective energy storage during periods of weight gain.

Disclosure

P. Varshney: None. L.M. Guth: None. C.M. Anderson: None. A. Raajendiran: None. M.J. Watt: None. J.F. Horowitz: Research Support; Self; American Diabetes Association.

Funding

National Institutes of Health (R01DK077966, T32DK101357)
292-OR: Renin-Angiotensin System Inhibition, Cardiorespiratory Fitness, and Mortality Risk in Diabetic Patients
Authors: KOKKINOS PETER , MYERS JONATHAN N. , SIDOSSIS LABROS , FASELIS CHARLES , NARAYAN PUNEET , NYLEN ERIC , SUI XUEMEI , MOORE HANS , KARASIK PAMELA
Published: June 2019

Introduction: Patients with type 2 diabetes mellitus (T2DM) have significantly higher risk of cardiovascular events and mortality. Angiotensin-converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARBs) decrease mortality risk in T2DM patients. Cardiorespiratory fitness (CRF) is associated with lower mortality risk in T2DM patients. However, the combined effects of CRF and ACEI/ARBs on mortality risk in T2DM patients has not been assessed.

Methods: We identified 6,888 T2DM patients (age 58.3±10.1 years) with no evidence of ischemia as indicated by a maximal standardized exercise treadmill test. We established two CRF categories based on age-adjusted median peak metabolic equivalents (METs) achieved: Unfit (5.4±1.3 METs; n=3,474) and Fit (9.0±2.3 METs; n=3414). We then formed 2 groups within each CRF category (treated and not treated with ACEI/ARBs) for a total of four CRF-ACEI/ARBs groups. Cox proportional hazards models, adjusted for known risk factors were constructed to assess the CRF-ACEI/ARBs interaction and mortality risk association.

Results: During a follow-up of 14.2±6.9 years, 2,495 patients died, accounting for 25.5 events/1,000 person-years of observation. Patients treated with ACE/ARBs had 25% lower mortality risk compared to those not treated with ACE/ARBs (HR 0.75; 95% CI: 0.69-0.82). When CRF-ACEI/ARBs categories were considered, Unfit individuals not treated with ACEI/ARBs had 24% higher mortality risk compared to those treated with ACEI/ARBs (HR 1.24; 95% CI 1.11-1.38). For Fit individuals, the risk was 26% lower (HR: 0.74; CI: 0.66-0.83) for those not treated with ACEI/ARBs and 45% lower in those treated with ACEI/ARBs (HR 0.55; 95% CI 0.48-0.62).

Conclusions: ACEI/ARBs therapy and increased CRF are effective in lowering mortality risk in in T2DM patients. The combination of ACEI/ARBs and higher CRF was synergistic, resulting in substantially lower mortality risk than either therapy alone.

Disclosure

P. Kokkinos: None. J.N. Myers: None. L. Sidossis: None. C. Faselis: None. P. Narayan: Research Support; Self; AstraZeneca, Eli Lilly and Company. E. Nylen: None. X. Sui: None. H. Moore: None. P. Karasik: None.
299-LB: Endothelial Regulation of Pancreatic ß-Cell Homeostasis in Diabetes
Authors: LI MANNA , QIAN MING , XU JIAN
Published: June 2019

Diabetes is the result of having an inadequate supply of functional insulin-producing β-cells. Efforts in research and development that focused on using systemic pathway to induce β-cell regeneration have had limited clinical success. Emerging studies implicate local islet molecular and cellular factors in pancreatic β-cell homeostasis. Intraislet endothelial cells (IECs), a key ally of pancreatic β-cells, secrete factors that could affect β-cell survival and insulin production. Fibroblast growth factor (FGF) 21 is an endocrine hormone that regulates energy homeostasis and insulin sensitivity. The function of endogenous FGF-21 derived from ECs is undefined in diabetes. With a loss-of-function approach, this study sought to determine the role of EC-FGF-21 in pancreatic β-cell homeostasis in STZ-induced diabetic mice and high-fat diet (HFD)-induced obese mice. Mice with EC-specific deletion of FGF-21 gene (EC-FKO) were generated by crossing Tie2-Cre mice with FGF-21loxP mice. In 8-to-10-week-old male mice (n=15/group) treated with STZ (50 mg/kg, i.p. for consecutive 5d and killed 4 weeks later), EC-FKO vs. wild type C57BL/6J (WT) mice displayed more severe hyperglycemia, degeneration of insulin-producing β-cells, and expansion of glucagon-producing α-cells. Both groups showed similar changes in pancreatic Glut2 and PARP1 protein levels and vascular density. In 8-to-10-week-old WT male mice (n=15/group) fed an HFD (TestDiet: 60% energy from fat) for 16 weeks, obesity and insulin resistance developed, along with lipid accumulation in the islet and compensatory expansion of β-cell mass, all of which were worsened, except for less β-cell mass expansion, in EC-FKO mice. Our latest immunofluorescent data showed a significant reduction of IEC-FGF-21 protein staining in WT mice treated with STZ or HFD vs. non-treated mice. Our findings suggest that EC-FGF-21 is required to maintain β-cell homeostasis under stress conditions. Loss of IEC-FGF-21 may contribute, at least in part, to β-cell degeneration in diabetes.

Disclosure

M. Li: None. M. Qian: None. J. Xu: None.

Funding

National Institutes of Health
30-OR: Whole Genome Sequence Analysis of Type 2 Diabetes Risk in 44,713 Humans of Diverse Ancestry in the TOPMed Study
Authors: HIGHLAND HEATHER M. , WESSEL JENNIFER , MANNING ALISA ,   TOPMED DIABETES WORKING GROUP
Published: June 2019

Risk of developing type 2 diabetes (T2D) is heritable, with over 403 independent genetic variants having been identified primarily through analysis of common variants in European ancestry populations. To further illuminate the genetic architecture of T2D, we conducted a whole genome sequence (WGS) association study (>38x depth) of common, low frequency, and rare variants in 9,663 individuals with T2D and 35,050 controls of diverse ancestry from NHLBI’s Trans-Omics for Precision Medicine (TOPMed) study. We conducted single variant analyses and SKAT tests of rare variants (MAF <1%) annotated with pancreatic islet specific regulatory annotation to define variant sets. In single variant analyses, a total of 15 regions were significantly associated (α=5e-08) with T2D, seven of which are novel associations (in or near ODF2L, LMAN2, KCNV1, CBR1, VLDLR-AS1, LINC01052 and NKX2-5). These new associations were either ancestry specific or rare variants. For instance, a variant not present in other ancestral groups, near KCNV1, was associated with increased risk of T2D (MAF 5.5%, P= 1.57e-08, OR=1.43) in African ancestry. Set based testing of rare variants identified seven regions associated with T2D in at least one ancestry. Six of these were noncoding: in African ancestry CBR1 gene centric variants (cumulative MAC[cMAC]=623, P = 5.85e-09) and an active promoter region in proximity to MRPL46 and MRPS11 (cMAC=3,218, P = 4.89e-07); and in Asian ancestry an enhancer hub containing NXPH3 (cMAC=3,317, P = 3.42e-05) and an active enhancer region linked to RP11-135A1.2 and HES1 (cMAC=57, P = 4.87e-07) were associated with T2D. KLRC4 predicted deleterious coding variants were associated with T2D in the European ancestry group (cMAC=25, P = 3.63e-06). In large scale WGS analyses, we extended our knowledge of the genetic basis of T2D through the inclusion of ancestrally diverse samples, variant typing without imputation, and use of tissue specific regulatory annotation to identify new T2D loci.

Disclosure

H.M. Highland: None. J. Wessel: Employee; Spouse/Partner; Eli Lilly and Company. A. Manning: None.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (5U01DK078616-10, K01DK107836)