bluuebunny/crossref_metadata_2025_split huggingface dataset:
1779-P: Characterization of Insulin-Regulated CLASP2 Phosphorylation
Authors: BARKER NATALIE K. , KRANTZ JAMES L. , LANGLAIS PAUL R. 
Published: June 2019

During an unbiased proteomics screen of rat L6 myotubes, we previously discovered the first member of the microtubule plus-end interacting protein (+TIP) family that undergoes insulin-stimulated phosphorylation, CLIP-associating protein 2 (CLASP2). Here we report that CLASP2 protein is expressed in several muscle groups as well as epididymal fat in mouse and we have successfully confirmed the expression of CLASP2 protein in human vastus lateralis muscle by mass spectrometry. In addition, insulin resistant, 60% fat diet-fed mice exhibit a loss of CLASP2 expression in epididymal adipose tissue versus control animals. We performed an insulin time course and discovered that insulin stimulates the phosphorylation of both endogenous and overexpressed CLASP2 in 3T3-L1 adipocytes in a manner to that similarly observed in L6 myotubes. Inhibition of either PI 3-kinase with wortmannin or mTOR with rapamycin had no effect on insulin-stimulated, proline-directed serine phosphorylation of CLASP2, whereas inhibition of the MAPK pathway reduced proline-directed serine phosphorylation of endogenous CLASP2 to basal levels. Quantitative phosphoproteomics identified Ser884/Ser1004/Ser1005/Ser1021 as significant insulin-stimulated CLASP2 phosphorylation sites that can be decreased after inhibition of the MAPK pathway. These findings indicate that CLASP2 undergoes insulin-stimulated proline-directed serine phosphorylation that is under control of the MAPK pathway in 3T3-L1 adipocytes. In addition, we also present new evidence of CLASP2 protein expression in mammalian tissues that are specifically targeted for insulin-stimulated glucose uptake and suggest that CLASP2 is altered within in vivo models of insulin resistance and type 2 diabetes.

Disclosure
N.K. Barker: None. J.L. Krantz: None. P.R. Langlais: None.

Funding
National Institutes of Health
1783-P: Translocon-Associated Protein Alpha (TRAPα) Is Required for Insulin Biosynthesis in ß Cells
Authors: LL XIN 
Published: June 2019

The discovery of missense mutations in the signal peptide of preproinsulin that causes congenital diabetes highlights the critical role of ER translocation in insulin biosynthesis. However, the mechanisms that govern these early events in insulin biosynthesis are poorly understood. GWAS studies have shown that translocon-associated protein alpha (TRAPα) is a high risk allele for both type 2 diabetes and gestational diabetes. Yet, the pathophysiological role of TRAPα in β-cell function remains unknown. Here we show that TRAPα is the most upregulated subunit of the TRAP complexes in isolated islets exposed to high glucose, suggesting that it may play an important role in insulin biosynthesis. Crispr/cas9 mediated TRAPα deletion in pancreatic INS832/13 cells causes inefficient preproinsulin translocation across the ER membrane, resulting in an accumulation of un-translocated preproinsulin in the cells. Surprisingly, TRAPα deletion also affects proinsulin ER export, leading to a decreased insulin production in INS832/13 cells. Re-introducing an epitope-tagged TRAPα to TRAPα KO cells rescued preproinsulin translocation defect and restored insulin production in a dose-dependent manner in TRAPα KO cells infected with adenovirus encoding TRAPα. Importantly, TRAPα deletion did not significantly affect the biogenesis or secretion of α1-antitrypsin, indicating that TRAPα promotes the ER translocation and subsequent trafficking of only a subset of secreted proteins. These results establish preproinsulin as a client protein for TRAPα in ER translocation and unveil unanticipated function of TRAPα in proinsulin ER export and insulin biosynthesis, and provide key insights into the disease pathogenesis associated with TRAPα deficiency.

Disclosure
X. Ll: None.

Funding
National Natural Science Foundation of China (81570699, 81620108004, 81370895); National Institutes of Health (R01DK088856, DK111174)
1784-P: Studying the Impact of Heterozygous Human INS Gene Mutation on Pancreatic ß Cell
Authors: AMIRRUDDIN NUR SHABRINA , TAN YAW SING , VERMA CHANDRA S. , GARDNER DAPHNE , BEE YONG MONG , HOON SHAWN , TEO ADRIAN 
Published: June 2019

Neonatal diabetes affects 1 in 90,000-200,000 live births. Permanent neonatal diabetes mellitus (PNDM) presents itself in the first few months of life, and patients require treatment henceforth. PNDM can be caused by mutations in the insulin gene, INS, most of which are heterozygous. This study involves two INS gene mutations: C109Y and G32V. The first mutation was reported in a Singaporean family, and the latter has not been reported yet. Patients with either mutation are unable to secrete insulin despite having one wild type (WT) allele. We hypothesize that both C109Y and G32V mutations result in misfolding of the proinsulin protein, leading to β-cell dysfunction. The misfolded proinsulin is then able to exert its dominant negative effect on WT proinsulin, either directly through heteromerization, or indirectly through altering endoplasmic reticulum (ER) integrity, preventing insulin secretion and eventually insulin synthesis and/or even β-cell death. We computationally modelled both C109Y and G32V proinsulin structures, and found that both mutations result in conformational changes that likely affect further insulin processing. We also overexpressed C109Y and G32V preproinsulin in a mouse insulinoma cell line, MIN6. Analysis of electron microscopy images revealed differences in the size and number of insulin granules. In addition, we obtained skin fibroblasts from patients with the C109Y mutation and their family members, reprogrammed them into induced pluripotent stem cells (iPSCs), and characterized them. We differentiated the iPSCs into β-like cells and saw no difference in size between WT and mutant cells. Current efforts are underway to determine the impact of the mutant insulin gene in the iPSC-derived β-like cells. Our findings will reveal mechanistic insights into this neonatal diabetes condition caused by the mutant insulin gene and potentially identify therapeutic approaches to reinstate the function of the existing wild type insulin gene.

Disclosure
N. Amirruddin: None. Y. Tan: None. C.S. Verma: Other Relationship; Self; sinopsee therapeutics. D. Gardner: None. Y. Bee: None. S. Hoon: None. A. Teo: None.

Funding
Institute of Molecular and Cell Biology; Khoo Teck Puat Hospital; National University Health System; Agency for Science, Technology and Research; National Medical Research Council; Lee Foundation; Skin Research Institute of Singapore
1787-P: Protein Kinase C (PKC) Negatively Regulate Neuronal Insulin Signaling In Vitro
Authors: MISHRA DEVANSHI , DEY CHINMOY S. 
Published: June 2019

PKCs are serine/threonine kinases sub-divided into three categories, namely, conventional, novel and atypical. Depending upon the isoform some have been reported to regulate insulin signaling positively and some negatively, in peripheral insulin responsive tissues like, skeletal muscles, adipocytes and hepatocytes through PI3K-Akt pathway, leading to glucose uptake. Though, peripheral tissues have been extensively studied, neuronal insulin signaling is less explored. Neuronal insulin signaling is required for maintaining cognitive abilities including synaptic plasticity and memory formation. Therefore, it is important to explore the role of PKC in neuronal insulin signaling. The objective of the present study was to elucidate the role of PKC in regulating insulin signaling in neuronal cells in vitro. To study that, differentiated Neuro-2a (N2a) cells were cultured in vitro and treated with or without phorbol 12-myristate 13-acetate (PMA), a well-known activator of PKC, with or without insulin stimulation and the effect was tested on the insulin signaling proteins. PKC when activated by PMA (400nM) and stimulated with insulin (100nM), down-regulated the activity of Akt by 25% (serine-473) and 20% (threonine-308). Testing of further downstream insulin signaling molecules under the same conditions demonstrated decrease in activity of AS160 by 20% and GSK3β by 20%. However, there was no change in the activity of GSK3α, suggesting a specificity of PKC towards GSK3β in neurons. To determine the functionality of these phosphorylations/activations, glucose uptake measurement showed that PKC decreased glucose uptake by 25%. Taken together these data strongly demonstrates for the first time that PKC negatively regulates insulin signaling cascade via Akt-AS160-GSK3β-glucose uptake pathway in neuronal cells. Further knowledge in this regard will pave the way for future understanding of insulin signaling in neuronal system and possibly neurodegenerative disorders.

Disclosure
D. Mishra: None. C.S. Dey: None.

Funding
Council of Scientific and Industrial Research, Government of India, New Delhi; Department of Science and Technology (SR/SO/HS-238/2012, SR/S2/JCB-24/2008(G))
1790-P: Akt2 Is Required for Glucose Uptake in Neuronal Insulin Signaling In Vitro
Authors: SHARMA MEDHA , DEY CHINMOY S. 
Published: June 2019

Akt2, an important protein kinase of insulin signal transduction pathway has been linked to regulating glucose homeostasis in insulin sensitive tissues like skeletal muscles, adipocytes, and hepatocytes. Any deregulation in its activation leads to a diabetic phenotype in peripheral cellular systems. Neuronal insulin signaling is associated with memory formation and synaptic plasticity, with Akt2 contributing to neuronal maturation and outgrowth, polarization and axon branching, and survival. Impaired neuronal signaling is also linked to neurodegenerative disorders like Alzheimer's disease (AD), which is increasingly being recognized as a brain-specific form of diabetes. However, previous studies on the role of Akt2 in neuronal cells are extremely limited. Thus, the aim of this study was to investigate the role of Akt2 in regulating neuronal insulin signaling. Akt2 was silenced using Akt2 specific siRNA or scrambled siRNA (control) in differentiated Neuro 2a (N2a) cells and the effect was studied on downstream signaling molecules of insulin signaling as well as on glucose uptake in presence or absence of insulin. Akt2 silencing caused a significant decrease (26% and 24%) in phoshorylation, and hence activation, of both, GSK3β and AS160 respectively, without affecting their expression. Akt2 silencing also resulted in significant decrease (30%) in glucose uptake when compared to corresponding silenced control in presence of insulin. Our data demonstrates for the first time that in neuronal cells Akt2 positively regulates insulin signaling via Akt2-AS160-GLUT4 arm. Data that suggests Akt2 plays a quintessential role in insulin signaling and glucose uptake even in a neuronal system is significant in terms of further understanding of neuronal insulin signaling and therefore possibly neuronal insulin resistance and neurodegenerative disorders.

Disclosure
M. Sharma: None. C.S. Dey: None.

Funding
Council of Scientific and Industrial Research, Government of India, New Delhi; Department of Science and Technology, Government of India, New Delhi (SR/S2/JCB-24/2008(G))
1792-P: Circumventing Gut Exposure to Bromocriptine (b) Administration Multiplies Its Antidiabetes Efficacy vs. Its Equimolar Plasma Level Derived from Oral Dosing
Authors: EZROKHI MICHAEL , LUO SHUQIN , CINCOTTA ANTHONY H. 
Published: June 2019

The antidiabetes drug, bromocriptine-QR is formulated to produce a brief pulse of dopaminergic (D) activity within the brain when taken orally within 2 hours of waking in an effort to mimic/re-establish the natural circadian peak of brain D activity observed in insulin (I) sensitive states. However, oral B is subject to extensive first pass metabolite (M) generation (&gt; 20 Ms) that can enter the circulation and influence catecholamine receptors. Also, B or B-M interactions with the gut can potentially influence (via afferents) brain metabolism centers. As such, gut/liver influences on B may limit the full antidiabetes impact of plasma B entering the brain to briefly activate dopamine receptors shortly after its oral dosing. To test this postulate, separate groups of I resistant, glucose (G) intolerant hamsters were dosed B daily at waking for 2 weeks either intraperitoneally (ip) at its established EDmax (4 mg/kg BW) or orally (16-28 mg/kg BW) (N=6/gr) so as to produce an equivalent plasma Cmax (20 ng/ml) and bioavailability (AUC180- 2000 ng*min/ml) of B and then subjected to an ip glucose tolerance test (GTT) along with controls (N=6). Relative to control, oral B did not alter fasting plasma G (FPG), FPI, GTT G or I, or I sensitivity (HOMA-IR or Matsuda Index) however, ip B did reduce FPG, FPI, GTT G and I by 40% (P&lt;0.02), 59% (P&lt;0.003), 45% (P&lt;0.002), and 54% (P&lt;0.03) respectively and improve HOMA-IR and Matsuda Index by 76 and 264%; P&lt;0.008 (and by an additional 73% and 577% respectively,[P&lt;0.002]) relative to oral B) even though each administration routine produced the same plasma Cmax and bioavailability of B. The ip B also reduced body fat by 29% (P&lt;0.006) without altering food intake, whereas oral B was without effect. These findings support the postulate that eliminating first pass B exposure to the gut can markedly enhance the antidiabetes efficacy of B and may offer a simple means of doing so for bromocriptine-QR in humans.

Disclosure
M. Ezrokhi: Employee; Self; VeroScience LLC. S. Luo: Employee; Self; VeroScience LLC. A.H. Cincotta: Employee; Self; VeroScience LLC.
1799-P: Lipotoxicity Impairs Central Regulation of Endogenous Glucose Production (EGP) in Type 2 Diabetes (T2D)
Authors: ALEKSIC SANDRA , ZHANG KEHAO , YIMAGOU ERIC LONTCHI , SHARMA SHIKSHA , TOMUTA NORICA , KEHLENBRINK SYLVIA , KISHORE PREETI , STEIN DANIEL T. , ROUTH VANESSA H. , HAWKINS MEREDITH 
Published: June 2019

Brain signals are integral to maintaining normal glucose homeostasis. Activation of hypothalamic KATP channels with diazoxide (DZX) reduces EGP in nondiabetic rodents and humans, but not in T2D. High fatty acids levels, typical of T2D, can increase endoplasmic reticulum (ER) stress and thereby impair glucose sensing by hypothalamic neurons.
We thus hypothesized that lowering fatty acids with nicotinic acid (NA) could restore suppression of EGP by central KATP channels in T2D. We performed 4-h euglycemic 'pancreatic clamp' studies (somatostatin with replacement of basal insulin, glucagon and growth hormone) in n=15 T2D subjects (11M, 50±2 years, BMI=30±1 kg/m2, HbA1c=9.7±0.5%) under 4 conditions: placebo (PL) vs. DZX (6 mg/kg, n=8) vs. DZX with 16-h NA infusion (DZX+NA; 0.01 mg/kg/min; n=5) vs. NA alone (n=7) (Figure 1a). DZX failed to suppress EGP; the response of EGP to DZX was restored by adding NA (Figure 1b). Confirming our hypothesis that diabetes impacts hypothalamic ER stress and KATP channels, several markers of ER stress were increased while expression of the Kir6.2 subunit of the KATP channel trended lower in hypothalamic wedges of Zucker Diabetic Fatty (ZDF) vs. nondiabetic rats (Figure 1c).
Hypothalamic ER stress and altered KATP channel expression may contribute to the loss of central regulation of EGP in T2D. Regulation of EGP may be normalized by reducing fatty acid levels, thus improving glycemic control.

Disclosure
S. Aleksic: None. K. Zhang: None. E. Lontchi Yimagou: None. S. Sharma: None. N. Tomuta: None. S. Kehlenbrink: None. P. Kishore: None. D.T. Stein: None. V.H. Routh: None. M. Hawkins: Other Relationship; Self; Novo Nordisk Inc.

Funding
National Institutes of Health (R01DK069861)
1804-P: Characterizing Central Olfactory Circuits Modulating Energy Homeostasis
Authors: RIERA CELINE 
Published: June 2019

Olfactory inputs are important for hedonic evaluation of food, resulting in food choice and possible consumption. Olfactory acuity is determined by the feeding status, with hunger enhancing smell and food seeking behavior, whereas satiety suppresses olfaction and promotes energy expenditure. Efforts to understand the cellular basis of food intake and energy expenditure revealed the crucial role of the hypothalamus as a master regulator of whole-body energy homeostasis, integrating internal and external stimuli to modulate energy intake and expenditure accordingly. However, how the hypothalamus adjusts circuits regulating energy homeostasis depending on external stimuli, such as smell, remains an intriguing mystery. Our previous work revealed that ablation of olfactory sensory neurons (OSNs) stimulates hypothalamic-driven autonomic tone and promotes catabolic pathways, enhancing thermogenesis in adipose tissue. These findings unravel a new function for the olfactory system in controlling energy homeostasis in response to sensory signals. Here, we analyzed neural circuits connecting olfaction and hypothalamus and determined the role of olfactory inputs on energy homeostasis. To identify neural circuits linking olfactory and hypothalamic neurons, we conducted anterograde viral tracing from mitral cell layer in the main olfactory bulb (MOB) in Tbx21-Cre mice and retrograde viral tracing from pro-opiomelanoctin (POMC)- and agouti-related protein (AgRP)-expressing hypothalamic neurons in POMC-Cre and AgRP-IRES-Cre mice using Cre-dependent helper virus and modified rabies virus. Additionally, we used chemical ablation of mature OSNs and chemogenetic silencing of mitral cell layer neurons to determine the effect of olfaction on energy homeostasis. Our data suggest that piriform cortex (PC)-amygdala (Amy)-arcuate nucleus of hypothalamus (ARC) is one of the plausible circuits responsible for transferring signals from MOB to the hypothalamus.

Disclosure
C. Riera: None.

Funding
American Diabetes Association/Pathway to Stop Diabetes (1-15-INI-12)
1805-P: Insulin Regulates a Broad Network of Gene Expression in the Brain to Regulated Brain Metabolism and Neurotransmission
Authors: CAI WEIKANG , BATISTA THIAGO M. , MARTIN RUBEN GARCIA , RAMIREZ ALFRED , KONISHI MASAHIRO , BRIAN T. O'NEILL , KIM JASON K. , KAHN C. RONALD 
Published: June 2019

Impaired insulin signaling in brain leads to both metabolic and behavioral abnormalities. To understand the role of insulin on gene expression in the brain, we performed hyperinsulinemic-euglycemic clamps using physiological doses of insulin in C57BL/6 mice, and after 3 hours collected different brain regions and analyzed their transcriptomes by RNA Seq. In response to peripheral insulin, 1851 genes were up- or down-regulated in the hypothalamus by at least 50% (all P &lt; 0.01). Other regions showed different responses with 205 genes significantly regulated by ≥ 50% in hippocampus and 131 regulated in nucleus accumbens. Insulin upregulated the pathway for very long chain fatty acyl-CoA synthesis in all three regions, but most insulin responsive genes were region specific. Thus, in hypothalamus, insulin modulated metabolism by suppressing the glycolysis and pentose phosphate pathway, while increasing expression of genes in the pyruvate dehydrogenase complex and cholesterol biosynthesis. Conversely, mice with streptozotocin diabetes displayed decreased expression of genes involved in the regulation of pyruvate dehydrogenase complex, very long-chain fatty acyl-CoA and cholesterol biosynthesis in hypothalamus, all of which were normalized by exogenous insulin. In addition, insulin regulated many genes in the hypothalamus involved in neurotransmission, including enhancing the expression of multiple GABA-A receptor subunits and suppressing many neuropeptides. Based on the transcriptional signature, changes in gene expression by insulin involve neurons, astrocytes, oligodendrocytes, microglia and endothelial cells.
In summary, peripherally administered insulin rapidly and potently regulates expression of genes involved in neurotransmission, neuromodulation and brain metabolism, especially in the hypothalamus. This helps reroute substrate carbons to the biogenesis of plasma membrane for neuronal and glial function and synaptic remodeling.

Disclosure
W. Cai: None. T.M. Batista: None. R. Garcia Martin: None. A. Ramirez: None. M. Konishi: None. B.T. O'Neill: None. J.K. Kim: None. C. Kahn: Advisory Panel; Self; MedImmune. Board Member; Self; Kaleido Biosciences. Consultant; Self; AntriaBio, Inc., Cobalt Therapeutics, Flagship Pioneering. Research Support; Self; Alnylam.
1815-P: Insulin Sensitivity Predicts Cognitive Decline in Prediabetes
Authors: WILLMANN CAROLINE , BROCKMANN KATHRIN , WAGNER ROBERT , KULLMANN STEPHANIE , PREISSL HUBERT , MAETZLER WALTER , GASSER THOMAS , ESCHWEILER GERHARD W. , METZGER FLORIAN G. , FALLHGATTER ANDREAS J. , HAERING HANS-ULRICH , FRITSCHE ANDREAS , HENI MARTIN 
Published: June 2019

The link between type 2 diabetes and cognitive dysfunction appears to start in the prediabetic state. Although cross sectional studies have linked insulin resistance to impaired cognition, this mechanism has not yet been sufficiently studied longitudinally without confounding by overt diabetes (and its treatment). We investigated participants of the ‘Tübinger Evaluation of Risk Factors for Early Detection of Neurodegeneration’ (TREND) study longitudinally. Subjects underwent a comprehensive neurocognitive assessment battery at baseline and every 2 years afterwards (median follow-up 4.0 q1-3: 2.2-4.3 y). Subjects with an HbA1c of 5.6-6.5% without known diabetes were metabolically investigated by a five-point 75g oral glucose tolerance test (OGTT) with assessment of insulin sensitivity and insulin secretion (N=175). Subjects with newly diagnosed diabetes or with depression (BDI &gt;20) were excluded (N=15). Data were analyzed by mixed models using sex, age and glycemic trait as fixed effects. Subject and time between measurements were used as random effects. Insulin sensitivity was negatively associated with the CERAD sum score (Consortium to Establish a Registry for Alzheimer's Disease) in a time-dependent manner (p=0.0057). This result is mainly driven by a worse performance in the memory domain due to declining insulin sensitivity (p=0.029). These interactions remained significant after adjusting for glycemia (p&lt;0.04). No interactions between time and insulin sensitivity for executive, visuospatial or language domains were found. There was no association with insulin secretion.
In summary, insulin resistance predicts cognitive decline, specifically memory impairment in prediabetic persons. Therefore, insulin resistance rather than sole elevation of blood glucose predispose neuro-cognitive impairment. Diabetes treatments that improve insulin sensitivity might therefore have the potential to postpone/prevent cognitive decline in diabetes.

Disclosure
C. Willmann: None. K. Brockmann: None. R. Wagner: Other Relationship; Self; Lilly Diabetes, Novo Nordisk A/S. S. Kullmann: None. H. Preissl: None. W. Maetzler: None. T. Gasser: Research Support; Self; National Institute on Aging. Other Relationship; Self; AbbVie Inc. G.W. Eschweiler: None. F.G. Metzger: None. A.J. Fallhgatter: None. H. Haering: None. A. Fritsche: None. M. Heni: Research Support; Self; Boehringer Ingelheim International GmbH, Sanofi. Speaker's Bureau; Self; Lilly Diabetes, Merck Sharp & Dohme Corp., Sanofi.