bluuebunny/crossref_metadata_2025 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 (> 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<0.02), 59% (P<0.003), 45% (P<0.002), and 54% (P<0.03) respectively and improve HOMA-IR and Matsuda Index by 76 and 264%; P<0.008 (and by an additional 73% and 577% respectively,[P<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<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 < 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 >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<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.
1818-P: Brainstem GABAergic Circuits Regulating Energy Balance
Authors: NECTOW ALEXANDER R.
Published: June 2019

Obesity is a serious disorder for which there is currently no effective treatment. The hypothalamus has long been studied as the critical node for regulating food intake and body weight; however, no effective targets have yet been identified within the hypothalamus for anti-obesity therapy. There is thus a critical, unmet need to identify other loci within the central nervous system that: a) potently regulate energy balance, through alterations in food intake and energy expenditure, and b) are potential therapeutic targets. We have recently found that the brainstem’s dorsal raphe nucleus (DRN) houses a small population of inhibitory, GABAergic neurons (hereafter DRNVgat neurons), which potently regulates energy balance, and concomitantly body weight. This is accomplished through the direct regulation of both food intake and energy expenditure, the latter component of which is achieved through both behavioral and physiologic mechanisms. However, the circuit mechanism(s) through which these neurons regulate energy homeostasis are poorly understood.

In recent work, we made the unexpected finding that DRNVgat neurons project broadly throughout the brain to differentially regulate both food intake and energy expenditure. In particular, we have found that these neurons exhibit both ascending projections (to the bed nucleus of the stria terminalis, dorsomedial hypothalamus, and medial preoptic area), as well as descending projections to the raphe pallidus. This latter, descending projection forms part of a polysynaptic circuit anatomically linking DRNVgat neurons to brown fat. Using projection-specific optogenetic manipulations, we have found that these projections can all potently suppress thermogenesis, and differentially regulate food intake. Critically, these effects are scalable, with higher stimulation intensities leading to increasing effects on both feeding and thermogenesis. Together, this work uncovers a novel circuit mechanism through which DRNVgat neurons regulate energy balance.

Disclosure

A.R. Nectow: None.

Funding

American Diabetes Association/Pathway to Stop Diabetes (1-18-ACE-49, 1-18-IBS-148 to A.R.N.); Foundation for Prader-Willi Research; National Alliance for Research on Schizophrenia and Depression
1842-P: Enhanced TGF-ß1 Signaling in the Ducts of Cystic Fibrosis Pancreas Increases Basal Insulin Secretion by Islets
Authors: ROTTI PAVANA GURURAJ , APAK-EVANS IDIL , OMALLEY YUNXIA , NORRIS ANDREW , UC ALIYE , ENGELHARDT JOHN
Published: June 2019

Cystic fibrosis (CF) is a multi-organ disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR protein is most highly expressed in pancreatic ductal epithelium, where loss of its function leads to ductal plugging, acinar destruction, and eventual diabetes. We hypothesize that exocrine pancreas remodeling in CF alters ductal paracrine signals to the endocrine pancreas. To test this hypothesis, we established primary ductal epithelium grown at an air-liquid interface (ALI) from WT and CFTR-KO ferret pancreas. Carbachol or IBMX/Forskolin (I/F) stimulated basolateral and apical secretions were analyzed for differentially secreted proteins using quantitative mass spectroscopy. Upon carbachol secretion 39 apical and 15 basolateral proteins were differentially secreted (P<0.05) by CFTR-KO ducts. Similarly, 26 and 6 differentially secreted proteins (P<0.05) were identified from I/F stimulated apical and basolateral secretions, respectively. TGF-β1 was one of the top upstream regulators of the differentially secreted proteins, P=5.02E-13 by Ingenuity Pathway Analysis. Single molecule fluorescence in situ hybridization of TGF-β1 mRNA on CFTR-KO and WT ferret pancreas indicated increased expression in CFTR-KO ducts compared to age matched controls. Likewise, expression of IGFBP7, a TGF-β1 signaling target, was augmented in fibroblasts surrounding pancreatic ducts and also in endocrine cells in CFTR-KO pancreas relative to controls. In cultured WT ferret islets, IGFBP7 treatment enhanced basal insulin secretion (2-fold, P<0.05) and attenuated glucose stimulated insulin secretion. This IGFBP7-dependent insulin secretion phenotype was similar to that observed in cultured CFTR-KO islets, suggesting TGF-β1 signaling may be partly responsible for altered insulin secretion in CF.

Disclosure

P. Rotti: None. I. Apak-Evans: None. Y. OMalley: None. A. Norris: None. A. Uc: None. J. Engelhardt: None.

Funding

National Institutes of Health
1846-P: The Insulin-Sensitizing Effects of Palmitic Acid Esters of Hydroxy Stearic Acids Involve Intertissue Communication
Authors: ZHOU PENG , SANTORO ANNA , PERONI ODILE D. , NELSON ANDREW T. , SAGHATELIAN ALAN , SIEGEL DIONICIO , KAHN BARBARA
Published: June 2019

Palmitic Acid esters of Hydroxy Stearic Acids (PAHSAs), bioactive lipids with anti-inflammatory and antidiabetic effects, improve glucose tolerance and insulin sensitivity in insulin-resistant mice. We found that PAHSA treatment enhances insulin action to suppress endogenous glucose production (EGP) in chow and HFD mice and promotes glucose uptake in glycolytic muscle and heart in HFD-fed mice. We aimed to determine the mechanisms by which PAHSAs enhance hepatic insulin sensitivity. We examined both direct effects in hepatocytes and indirect effects resulting from augmentation of insulin-mediated suppression of WAT lipolysis. Here, we show that PAHSAs inhibit lipolysis in WAT explants to the same extent as insulin. PAHSAs also enhance the anti-lipolytic effect of insulin in vivo, as indicated by a 30% reduction in FFAs during the clamp in PAHSA-treated mice under conditions with no suppression in Vehicle treated mice. To determine whether lowering FFAs is necessary for PAHSA effects on insulin sensitivity, we infused intralipid during a hyperinsulinemic-euglycemic clamp. In chow-fed mice infused with intralipid, PAHSAs still have a partial effect to lower EGP (Intralipid: vehicle 12±0.8 vs. PAHSA 7±1.7 mg/kg/minute), whereas in HFD-fed mice, preventing PAHSA-induced FFA reduction blocks PAHSA effects on hepatic insulin sensitivity (EGP Intralipid: vehicle 13±0.6 vs. PAHSA 12±1.7 mg/kg/minute). Our mechanistic studies show that PAHSAs inhibit basal EGP and reduce glucagon stimulated EGP directly in isolated hepatocytes. This is mediated by a cAMP-dependent pathway involving Gα/i protein-coupled receptors. Hepatic phospho-CREB is also reduced by PAHSAs in vivo. Sum: PAHSAs enhance hepatic insulin sensitivity through direct and indirect actions involving inter-tissue communication between adipose tissue and liver. These data reveal new mechanisms underlying the beneficial effects of PAHSAs, which will clarify their roles in healthy and disease states.

Disclosure

P. Zhou: None. A. Santoro: None. O.D. Peroni: None. A.T. Nelson: None. A. Saghatelian: None. D. Siegel: None. B. Kahn: Advisory Panel; Self; Alterna Therapeutics, Inc., American Diabetes Association, Harrington Discovery Institute, Janssen Research & Development. Consultant; Self; Ironwood Pharmaceuticals, Inc. Research Support; Self; National Institute of Diabetes and Digestive and Kidney Diseases.

Funding

American Diabetes Association (1-18-PDF-134 to P.Z.); National Institutes of Health (R01DK43051 to B.K.), (R01DK106210 to B.K., A.S.); JPB Foundation (to B.K.); American Heart Association (to A.T.N.)
1851-P: Bromocriptine (B) plus Curcumin (C) Interact to Reduce Advanced Fibrosis in a Mouse Methionine-Choline Deficient (MCD) Diet Model of Nonalcoholic Steatohepatitis (NASH)
Authors: TSAI TSUNG-HUANG , CINCOTTA ANTHONY
Published: June 2019

NASH is characterized by severe hepatic fibrosis that potentiates cirrhosis and hepatocellular carcinoma. The MCD diet also potentiates severe liver fibrosis by reducing lipid secretion and antioxidant defenses, and adversely altering the gut microbiome to promote liver inflammation and damage. B, a dopamine D2 receptor agonist reduces adipose lipolysis and hepatic lipogenesis and inflammation. C improves a gut microbiome adverse to liver. C is also known to augment brain dopaminergic activity. We therefore investigated if B and C combination therapy that engages different pathological targets of NASH may provide an additive/synergistic benefit against MCD diet induced liver fibrosis. C57B6 mice were fed an MCD diet for 8 weeks to induce NASH with fibrosis then randomized to 4 treatment groups: Vehicle, B (10mg/kg), C (100mg/kg), and combination of B and C (BC) daily for 4 weeks while maintained on the diet. A group of mice on normal diet were used as normal reference control. BC therapy led to significant reductions of serum ALT (85%), serum AST (33.4%), liver steatosis (65.4%), inflammation (86.7%), ballooning degeneration (60%), and NAFLD activity score (86.4%), as well as gene expression markers each of fibrosis (αSMA, TGFβ, COL1A, TXNIP) by 88-92%, inflammation (TNFa, IL1β, MCP1, iNOS) by 91-97%, ER stress (DDIT3, ATF, sXBP1, HSPA5) by 45-93%, steatosis (ACC1, PLIN2) by 31-97%, and fatty acid oxidation (PPARα, PGC1α, CPT1, UCP1) by 65-86, and reduced the percent fibrosis area by 92.7% relative to vehicle (and resultantly similar to normal diet mice) (all above: P<0.05) and by amounts greater than B or C alone or additively. This study demonstrates the positive interactive effects of bromocriptine and curcumin on advanced liver fibrosis in an MCD diet model of NASH.

Disclosure

T. Tsai: Employee; Self; VeroScience LLC. A. Cincotta: Employee; Self; VeroScience LLC.
1858-P: Bidirectional Relationship between Chronic Kidney Disease and Nonalcoholic Fatty Liver Disease in Patients with Type 2 Diabetes Mellitus
Authors: HSU HUI-CHUN , CHANG YU HUNG , AN LINGWANG , LEE TAO-CHUN
Published: June 2019

Background: Nonalcoholic fatty liver disease (NAFLD) and chronic kidney disease (CKD) are highly prevalent in type 2 diabetes mellitus (T2DM). Growing evidence suggests that NAFLD and CKD share common traditional risk factors, but it would seem unique risk pathways are involved as a consequence of NAFLD and CKD, efforts are still needed for a more comprehensive understanding of these relationship.

Methods: A structural equation model (SEM) was applied to assess associations between demographic data, inflammation factor, anthropometric and metabolic variables simultaneously and estimated glomerular filtration rate (eGFR) and fatty liver index (FLI). Given the potential that the NAFLD and CKD relationship may be bidirectional, SEMs also to examine direct and indirect effects of NAFLD on CKD, and vice versa.

Results: A total of 1,992 subjects with T2DM were enrolled in this study. Using multivariate analysis, NAFLD was independently associated with the risk of CKD (adjusted OR=1.59, 95% CI=1.12-2.25, P=0.009). SEMs showed that age (-0.216), triglyceride (-0.129), uric acid (UA, -0.415), albumin (0.163), and HbA1c (-0.066) levels had direct effects on eGFR, and the final model could explain 30% of the variability in eGFR. Age (0.094), triglyceride (0.174), body mass index (BMI) (0.707), UA (0.092), white blood cell (WBC) count (0.070), serum glutamic pyruvic transaminase (0.132) levels, and smoking (0.066) status had direct effects on FLI, and the final model could explain 67% of the variability in FLI. The common risk factors contributed to both eGFR and FLI were age, triglyceride, and UA. Unique risk factors were albumin and HbA1c for eGFR, and BMI, WBC, SGPT, and smoking for FLI. SEMs analysis confirm the bidirectional cause-relationships between NAFLD and CKD.

Conclusions: Common and unique risk factors and bidirectional relationship exist between CKD and NAFLD in patients with T2DM.

Disclosure

H. Hsu: None. Y. Chang: None. L. An: None. T. Lee: None.
1861-P: Pioglitazone Modulates Hepatic Pyruvate Dehydrogenase and TCA Cycle Flux
Authors: SHANNON CHRIS , RAGAVAN MUKUNDAN , DEFRONZO RALPH A. , MERRITT MATTHEW , NORTON LUKE
Published: June 2019

Background: The insulin sensitizing actions of the antidiabetic drug Pioglitazone (PIO) have classically been ascribed to the reversal of ectopic lipid accumulation. We recently demonstrated acute, direct metabolic effects of PIO in hepatocytes, linking the suppression of hepatic glucose production to an inhibition of mitochondrial pyruvate metabolism. As the relevance of this mechanism in vivo is unclear, we studied the effects of PIO on hepatic pyruvate metabolism in a mouse model of insulin resistance.

Methods: Eight-week-old male ob/ob mice were placed on a regular diet (10% kcal fat) with (n=10) or without (n=10) 300 mg/kg PIO for four weeks before study during euglycemic-hyperinsulinemic clamp (5 mU/kg min). Endogenous glucose production (EGP), hepatic glycogen synthesis and anabolic/oxidative pyruvate flux ratio (PC/PDH) were determined from [3-3H] glucose infusion and 13C NMR isotopomer analysis using [2,3-13C] pyruvate. Livers were freeze-clamped in situ for pyruvate dehydrogenase (PDH), metabolite and protein analysis.

Results: PIO treatment lowered EGP by 62% (4.6 ± 1.0 vs. 12.1 ± 1.7 mg/kg min; P<0.01) and increased glycogen synthesis 10-fold (19.7 ± 4.8 vs. 1.9 ± 0.8 μ mol/kg liver min; P<0.005), whereas PC/PDH remained unchanged. PIO also suppressed liver PDH activation (14.6 ± 3.4 vs. 29.6 ± 4.3 µmol/kg liver min; P<0.05), as well as acetylcarnitine (140 ± 18 vs. 297 ± 40) and acetyl-CoA (17.7 ± 2.4 vs. 47.8 ± 8.8 µmol/kg liver) concentrations (P<0.01), whilst liver PDK4 protein expression was increased 46% compared to controls (P<0.05).

Conclusions: Inhibition of PDH activation by Pioglitazone curtails mitochondrial acetyl-CoA delivery, relieving allosteric stimulation of pyruvate carboxylase and gluconeogenesis. Parallel reductions in PDH flux and EGP reconcile an unchanged PC/PDH ratio, suggesting an overall slowing of TCA cycle flux with PIO. These novel data highlight the therapeutic relevance of hepatic mitochondrial metabolism in the treatment of type 2 diabetes and liver disease.

Disclosure

C. Shannon: None. M. Ragavan: None. R.A. DeFronzo: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Elcelyx Therapeutics, Inc., Intarcia Therapeutics, Inc., Janssen Pharmaceuticals, Inc., Novo Nordisk Inc. Research Support; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc. Speaker's Bureau; Self; AstraZeneca, Novo Nordisk Inc. M. Merritt: None. L. Norton: None.

Funding

University of Florida’s Southeast Center for Integrated Metabolomics (U24DK097209); National Institutes of Health
1865-P: Dual Chemokine Receptor CCR2/CCR5 Antagonist Cenicriviroc Ameliorates Lipotoxicity-Induced Insulin Resistance and NASH in Mice
Authors: OTA TSUGUHITO , CHEN GUANLIANG , NAGASHIMADA MAYUMI
Published: June 2019

Hepatic lipid accumulation drives innate immunity with recruitment of bone marrow-derived macrophages and activation of liver-resident Kupffer cells, leading to insulin resistance and nonalcoholic steatohepatitis (NASH) development. However, treatment options that target macrophage/Kupffer cells recruitment or activation to halt the insulin resistance and NASH remain limited. A C-C chemokine receptor 2 (CCR2) plays a central role in macrophage recruitment. In addition, we reported that a different C-C chemokine receptor, CCR5, promotes insulin resistance and hepatic steatosis by regulating macrophage M1/M2 status. Here, we investigated the effect of cenicriviroc (CVC), a dual CCR2 and CCR5 receptor antagonist in a lipotoxic model of NASH with hepatic fibrosis and insulin resistance. Eight-week-old C57BL/6 mice were fed a high-cholesterol, high-fat (CL) diet or a CL diet containing 0.015% CVC (CL+CVC) for 12 weeks. The liver histology, insulin sensitivity, and fibrogenesis were examined. The CVC administration decreased the increase in plasma ALT levels as well as liver triglyceride, cholesterol, and TBARS levels caused by the CL diet. In addition, CVC improved glucose intolerance and hyperinsulinemia in the CL group and augmented the insulin signal, assessed by IRβ and Akt phosphorylation in the liver. Flow cytometry analysis revealed that mice fed CL+CVC had 72% fewer CD11c+CD206- M1-like macrophages, but 94% more CD11c-CD206+M2-like macrophages than CL group, indicating that CVC caused an M2-dominant shift in macrophages/Kupffer cells in the liver. Furthermore, CVC attenuated hepatic fibrosis by repressing the activation of hepatic stellate cells (HSCs) and decreasing hydroxyproline content. Thus, CVC ameliorated lipotoxicity-induced insulin resistance and NASH with hepatic fibrosis by polarizing M2 macrophage and attenuating HSC activation.

Disclosure

T. Ota: None. G. Chen: None. M. Nagashimada: None.

Funding

Japan Ministry of Education, Culture, Sports, Science and Technology
1866-P: Peripancreatic Adipose Tissue Protects against Ectopic Fat Accumulation in the Liver
Authors: CHANCLÓN BELÉN , MICALLEF PETER , NORDBECK ELIN BANKE , WU YANLING , WILDER BJÖRN , KANERVA JULIA , RORSMAN PATRIK ,   INGRID WERNSTEDT ASTERHOLM
Published: June 2019

Peripancreatic adipose tissue (PAT) is an understudied visceral fat depot that surrounds pancreas and is in touch with spleen and liver. Recent previous studies suggest that obesity-associated PAT inflammation accelerates pancreatic neoplasia in KrasG12D mice and that PAT conditioned-medium from pre-diabetic rats (9 weeks on high-fat diet; HFD) stimulates insulin secretion, proliferation and apoptosis in INS-1 cells. In this study, we aimed to better characterize PAT functionality in the context of HFD-induced obesity. PAT is a very small depot (0,5 ±0,04 mg) with a relatively high number of small adipocytes in unchallenged mice on chow, and we found that PAT expansion is less pronounced at early stages of HFD-feeding compared to the expansion of inguinal (IWAT), gonadal (GWAT) and mesenteric adipose tissues (MWAT). After 8-weeks HFD feeding the expansion of PAT was however comparable to the other three fat depots, but still PAT was more protected against the deleterious effects of HFD on adipose tissue inflammation and insulin resistance as judged by gene expression analysis (adiponectin, insulin receptor and inflammation markers). Feeding after 16-weeks HFD reduced the glucose and lipid uptake in all fat depots however, it was less pronounced in PAT; both fatty acid and glucose oxidation rates were higher in PAT compared to IWAT, GWAT and MWAT. Moreover, we noted that PAT is the only fat depot where the tissue weight correlates positively with liver weight in HFD-induced obese mice, and surgical removal of PAT followed by 16-week-HFD-feeding was associated with increased hepatic triglyceride content and accumulation of more and larger lipid droplets. PAT did not regenerate during this 16-week HFD time course suggesting that its precursor cells reside within the depot. Taken all data together, we propose that PAT, although it is located in the visceral compartment, can be considered as a metabolically/immunologically healthy fat depot that protects against hepatic steatosis in advanced obesity.

Disclosure

B. Chanclón: None. P. Micallef: None. E. Banke Nordbeck: None. Y. Wu: None. B. Wilder: None. J. Kanerva: None. P. Rorsman: None. I. Wernstedt Asterholm: None.
187-OR: Gut Microbiota Regulate Pancreatic Growth, Exocrine Function, and Gut Hormones
Authors: ALTINDIS EMRAH , SOTO MARION , ORLIAGUET LUCIE , HUANG QIAN , CEDERQUIST CARLY , DIRICE ERCUMENT , FUJISAKA SHIHO , HU JIANG , KULKARNI ROHIT , KAHN C. RONALD
Published: June 2019

Growing evidence suggests a link between alterations in gut microbiota, diabetes, and metabolic syndrome. We recently showed that treatment with antibiotics to alter gut microbiota could improve insulin signaling and glucose metabolism in obesity- and diabetes-prone C57BL/6J mice on high-fat diet (HFD). This occurred in part through changing metabolite composition in plasma and decreasing adipose tissue inflammation. We also showed that these changes could be induced in germ-free mice by transferring the gut microbiota. It is known that diabetes is associated with exocrine pancreatic dysfunction, and diseases of the exocrine pancreas can be associated with diabetes. To explore the role of gut microbiota on host pancreatic exocrine function and its crosstalk with the endocrine system, mice were given either normal chow or HFD, or HFD and treatment with vancomycin or metronidazole. As expected, HFD alone produced signs of metabolic syndrome but also increased total pancreas weight by ~20%, an amount that could not be explained by islet hyperplasia. Analysis of the fecal proteome of these mice identified 138 host proteins of which 32 changed with either HFD or antibiotic treatment. Notably, the most significantly changed proteins by diet and altered microbiome were pancreatic enzymes: amylase, lipase and elastase. Furthermore, the plasma levels of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) levels were decreased, while glucose-dependent insulinotropic peptide (GIP) was increased by the HFD. These alterations of pancreas growth, acinar cell hyperplasia, exocrine pancreas function, and hormone secretion were reversed by antibiotic treatment of the host. These changes could also be reproduced in germ free mice by transfer of gut microbiota. Together, these data indicate that gut microbiota can regulate pancreatic growth and both exocrine and endocrine function. Better understanding pancreas-gut microbiome crosstalk can help us develop probiotics to cure pancreas dysfunction in diabetes.

Disclosure

E. Altindis: None. M. Soto: Employee; Self; LNC Therapeutics. Other Relationship; Self; biomeostasis. L. Orliaguet: None. Q. Huang: None. C. Cederquist: None. E. Dirice: None. S. Fujisaka: None. J. Hu: None. R. Kulkarni: None. C. Kahn: Advisory Panel; Self; MedImmune. Board Member; Self; Kaleido Biosciences. Consultant; Self; AntriaBio, Inc., Cobalt Therapeutics, Flagship Pioneering. Research Support; Self; Alnylam.

Funding

National Institutes of Health
1875-P: Liver-Specific p38 MAPK Stimulates Hepatic Fat Accumulation and Reduces Insulin Sensitivity In Vivo
Authors: RIVERS SYDNEY L. , WANG HUIQI , LAM LORETTA , GIACCA ADRIA
Published: June 2019

Insulin resistance and hepatic fat accumulation are features of metabolic syndrome, although the mechanisms remain unclear. The cellular-stress responsive kinase, p38 Mitogen Activated Protein Kinase (p38), has controversial roles in regulating glucose and lipid metabolism in vivo. Here we show that liver-specific p38 stimulates hepatic triglyceride accumulation and contributes to the development of high fat diet (HFD)-induced insulin resistance. Liver-specific p38 knockout mice (p38KO) and wild type controls (WT) were fed a HFD or control diet for one week. Glucose tolerance tests revealed that HFD-fed p38KO mice had improved glucose tolerance compared to HFD-fed WT mice. Moreover, hyperinsulinemic-euglycemic clamps were performed to assess insulin sensitivity. HFD-fed p38KO mice were protected from reduced whole-body insulin action seen in HFD-fed WT mice, having similar glucose infusion rates as WT mice fed a control diet. To assess hepatic triglyceride accumulation, liver sections were stained with Oil Red-O post diet treatment. HFD-fed WT mice had significantly greater hepatic triglycerides, represented as a percentage of total liver area, compared to HFD-fed p38KO mice. In accordance with reduced hepatic triglyceride accumulation, decreasing trends in triglyceride secretion rates and plasma ApoB protein expression were reported between HFD-fed p38KO and HFD-fed WT mice following Poloxamer-407 injections. Overall, our data suggest that genetic inhibition of hepatic p38 may provide benefit in controlling heightened hepatic fat accumulation and reduced insulin sensitivity observed in HFD models.

Disclosure

S.L. Rivers: None. H. Wang: None. L. Lam: None. A. Giacca: Research Support; Self; Jazz Pharmaceuticals.

Funding

Canadian Institutes of Health Research