bluuebunny/crossref_metadata_2025 huggingface dataset:
203-LB: The Role of the Exocyst Trafficking Complex in CD36 Fatty Acid Translocase Trafficking in Skeletal Muscle Cells
Authors: NAKAMURA NICOLE K. , TOKUNAGA DARCY S. , HA HERENA Y.J. , POLGAR NOEMI
Published: May 2022

Skeletal muscle free fatty acid (FFA) uptake correlates with the amount of fatty acid translocase CD36 on the cell surface. In obesity, chronic membrane-localization of CD36, but not other FFA transporters, augments FFA uptake and intracellular lipid accumulation. This ectopic lipid accumulation then causes insulin resistance (IR) by inhibiting GLUT4 glucose transporter trafficking and glucose uptake in response to insulin. GLUT4 and CD36 cell-surface delivery is stimulated by insulin and contraction signaling, sharing conserved downstream effectors. The eight-protein exocyst complex is responsible for the targeted docking of membrane-bound vesicles, a process underlying cell-surface delivery of fuel transporters. The exocyst indeed regulates insulin-induced glucose uptake via GLUT4 trafficking to the membrane in adipocytes and skeletal muscle. In addition, it controls lipid uptake in adipocytes, but we do not know which of the FFA-transporting proteins the exocyst targets during this process. Based on the high degree of conservation of the fuel transporter trafficking mechanisms in adipose and skeletal muscle tissue, we hypothesized that the exocyst contributes to lipid uptake in skeletal muscle via the targeted trafficking of CD36. Our data shows that the exocyst plays a role in FFA uptake in skeletal muscle cells. Using an L6 GLUT4myc myoblast model we demonstrate that the exocyst is recruited to CD36 vesicles upon insulin and contraction signaling. We also show that disrupted exocyst activity leads to an impaired CD36 surface delivery and decreased FFA uptake in skeletal myoblasts. We are the first to demonstrate that the exocyst is necessary for insulin- and contraction-induced CD36 membrane trafficking and FFA metabolism in muscle cells. Further studies will continue to investigate the mechanism of exocyst-regulated FFA uptake and lipid metabolism in skeletal muscle to help our understanding of ectopic lipid accumulation leading to IR.

Disclosure

N. K. Nakamura: None. D. S. Tokunaga: None. H. Y. J. Ha: None. N. Polgar: None.
203-OR: VGLL4 and MENIN Function as TEAD1 Corepressors to Block Pancreatic ß-Cell Proliferation
Authors: LI FENG , LIU RUYA , NEGI VINNY , YANG PING , LEE JEONGKYUNG K. , MOULIK MOUSUMI , YECHOOR VIJAY
Published: May 2022

β-cell mass is significantly reduced especially in the late stage of diabetes. Hence, increasing β-cell proliferation to increase β-cell mass remains a desirable goal for diabetes treatment. The mechanisms that regulate β cell proliferation and concurrent maintenance of mature function is not well understood. Hippo-TEAD pathways regulated proliferation in many tissues. Our data show that TEAD1 (Hippo) pathway activities were significantly reduced in mature β cell. TEAD1 knockout results in the cell-autonomous increase of β cell proliferation, while loss of TAZ has not change in β cell proliferation, suggesting that TEAD1 corepressors, rather than coactivators, play a dominant role in β-cells. Improved Split-GFP system and yeast two-hybrid platform were also adapted to screen TEAD1 cofactors in β cells. Our study showed that MENIN can bind to TEAD1 directly and the pocket area of TEAD1 is critical for this binding. VGLL4 and MENIN work as TEAD1 corepressors to inhibit the expression of target genes in β-cells, including FZD7 and CCN2, which leads to a restriction of β cell proliferation. We also found that TEAD1 can regulate TEAD1, and this auto-inhibition contributes to TEAD1 pathway inhibition with β cell maturity. Our study sheds light on the mechanisms involved in β cell maintenance of mature function and proliferation and offer the mechanistic basis for modulating TEAD pathway to increase proliferation in β cells.

Disclosure

F.Li: None. R.Liu: None. V.Negi: None. P.Yang: None. J.K.Lee: None. M.Moulik: None. V.Yechoor: n/a.
212-LB: Decreasing Drp1 Expression in Mouse Liver Activates the Mitochondrial Integrated Stress Response and Exacerbates NASH
Authors: STEFFEN JANOS , NGO JENNIFER , WANG SHENG-PING , KRAMER HENNING F. , NORQUAY LISA D. , POCAI ALESSANDRO , SHIRIHAI ORIAN , LIESA MARC
Published: May 2022

Elevated Drp1-mediated mitochondrial fission was proposed to promote NAFLD, as inhibition of hepatic Drp1 in early life prevented high-fat diet induced liver steatosis in mice. However, whether Drp1-knockdown (Drp1si) can reverse established NASH in adulthood is unknown. Hepatocyte-restricted delivery of siRNA conjugated to N-acetyl-galactosamine (GalNac) is a safe and FDA-approved approach to selectively manipulate hepatic gene expression and treat human liver disease. Here, we show that hepatic Drp1si in adult healthy mice decreased body weight by 12% and induced liver damage, as shown by the elevation in circulating transaminases, liver inflammation, fibrosis and necrosis (p<0.001) . Decreased body weight was largely explained by Gdf15 actions decreasing food intake by 11%, as Gdf15 receptor (Gfral) KO prevented the decrease in food intake and mitigated the weight loss caused by Drp1-KD. An activation of the Atf4- integrated stress response (ISR) in liver explained the 4x fold increase in hepatic Gdf15 expression. Drp1si increased both ER stress and the activity of the mitochondrial protease Oma1, two processes that converge in increasing EIf2alpha phosphorylation and Atf4 expression. While NASH induced by GAN diet did not activate Oma1 by itself, Drp1si in mice with NASH was sufficient to activate Oma1 and exacerbate NASH-induced ER-stress. These led to higher ISR activation, exacerbated inflammation, fibrosis and necrosis.

In conclusion, our study supports that increased Drp1 activity is an adaptative mechanism counteracting NASH that prevents ISR overactivation and mitigates ER stress to limit fibrosis, inflammation, and necrosis. Our study argues against blocking Drp1 in hepatocytes to combat NASH.

Disclosure

J. Steffen: Employee; Janssen Research & Development, LLC. J. Ngo: None. S. Wang: None. H. F. Kramer: Employee; Janssen Research & Development, LLC. L. D. Norquay: Employee; Janssen Pharmaceuticals, Inc. A. Pocai: Employee; Johnson & Johnson. O. Shirihai: None. M. Liesa: None.
221-LB: Normalisation of Glycemic and Extraglycemic Parameters with T2DM Remission—Insights from RCT Enabled by Whole-Body Digital Twin Technology
Authors: SHAMANNA PARAMESH , SHAH LISA , MOHAMED MALUK , MOHAMMED JAHANGIR , POON TERRENCE , DHARMALINGAM MALA , VADAVI ARUN , SABOO BANSHI , DAMODHARAN SURESH , KESHAVAMURTHY ASHOK , THAJUDEEN MOHAMED , BHONSLEY SUCHITRA , JOSHI SHASHANK R.
Published: May 2022

We analyzed the efficacy of Twin Precision Treatment (TPT) : a novel whole-body digital twin enabled precision nutrition that measures weight, physical activity, sleep, and sensor glucose values, for achieving remission, at six months among 199 people with T2D enrolled in RCT. The mean age was 43 years (±8.7) ; diabetes duration was 3.7 years (±2.7) . 83.9% (167/199) achieved diabetes remission (HbA1c < 6.5, with the absence of glucose-lowering pharmacotherapy, for at least 3 months) . We looked at the change in the number and percentage of patients who were within the normal range for the biochemical, glycemic metrics, metabolic markers, and BMI, waist circumference, and ASCVD risk score (Figure) . There was a significant increase in the number of people having normal ranges at 90 and 180 days compared to baseline. The parameters for which ≥ 90% in the normal range at 180 days were A1C, FPG, GMI, CV, NAFLD FS, MICR, ASCVD risk score, and hsCRP. The proportion of normalisation rates were 60-90% for HOMA2B, HOMA2IR, BMI, waist circumference, TG/HDL, sdLDL, NAFLD LFS, TIR. Our study demonstrates the diverse metabolic benefits which can be obtained by whole-body digital twin enabled precision nutrition in addition to diabetes remission.

Disclosure

P. Shamanna: None. A. Keshavamurthy: None. M. Thajudeen: None. S. Bhonsley: None. S. R. Joshi: Advisory Panel; AstraZeneca, Consultant; Biocon, Franco India, Glenmark Pharmaceuticals, Marico, Sanofi, Twinhealth, Zydus Cadila, Speaker's Bureau; Abbott, Serdia Pharmaceuticals (India) Pvt. Ltd. L. Shah: None. M. Mohamed: None. J. Mohammed: None. T. Poon: None. M. Dharmalingam: None. A. Vadavi: None. B. Saboo: None. S. Damodharan: None.
223-LB: Interactive Effects of Dietary Iron and Chronic Hypoxia on Glucose Homeostasis in a T2D Mouse Model
Authors: HARRISON ALEXANDRIA V. , MCCLAIN DONALD A. , SINK SANDY T. , LORENZO FELIPE
Published: May 2022

The micronutrient iron is a risk factor for type II diabetes (T2D) . The effects of iron are, in part, mediated by the iron-sensing hypoxia inducible factor (HIF) pathway. The interactions of iron and hypoxia in the progression of T2D are not fully understood. Mice were fed a fast-food (FF) diet supplemented with either normal iron (35 mg/kg) or high iron (2000 mg/kg) under normoxia and hypoxia. Hypoxic mice had a significant reduction in weight compared to normoxic mice (by 16% for 35mg-Fe diet and 14% for 2000mg-Fe diet, p<0.001) . There was an interactive effect of iron and hypoxia on fasting glucose and glucose tolerance (p=0.016, p=0.41 respectively) in which the combination of hypoxia and normal iron yielded the best improvement. For insulin-mediated glucose uptake mechanisms, insulin sensitivity was observed with high iron and also under hypoxia when using the homeostatic model assessment (HOMA-IR) . Direct measurement using the insulin tolerance test showed no additional sensitivity was observed with hypoxia, suggesting non-insulin mediated glucose uptake (NIMGU) mechanisms. Indirect calorimetry demonstrates that hypoxia and iron interact to increase carbohydrate utilization and energy expenditure (p<0.001 and p=0.005, respectively) . Measured eWAT tissue mass showed significant reduction in tissue mass for hypoxic mice. Transcriptional profile of mouse eWAT shows significant upregulation of classic HIF target genes involved in glycolysis, mitochondrial metabolism, and intracellular iron homeostasis in hypoxic mice and interestingly also in normoxic high iron mice. Lastly, protein expression of HIF regulators FIH1 and PHD2 were significantly reduced (25-40% reduction, p<0.05 for FIH and p<0.01 for PHD2) under hypoxia. We conclude that the observed effects of dietary iron are dependent on oxygen levels, with the best protection against T2D offered by normal iron and hypoxia. Enhanced glucose disposal is due to NIMGU mechanisms, with the two HIF regulatory arms, PHD2 and FIH1, also mediating effects. Results from this study support novel targeted therapies and dietary changes to treat T2D.

Disclosure

A. V. Harrison: None. D. A. Mcclain: None. S. T. Sink: None. F. Lorenzo: None.

Funding

NIDDK Research Service of the Veterans Administration
226-OR: Reduced Fatty Acid Oxidation Prevents Intralipid-Induced Hepatic Insulin Resistance in Humans
Authors: PURNELL JONATHAN Q. , GREGOR ASHLEY N. , CHOI DONGSEOK , BAETSCHER ERIC , ROONEY WILLIAM , HARDING CARY O. , GILLINGHAM MELANIE B.
Published: May 2022

Mitochondrial handling of excess lipid substrate may be an important determinant of insulin resistance. Animal models support insulin signaling defects resulting from both defective fatty acid oxidation (FAO) with subsequent intra-cellular lipid accumulation on the one hand and enhanced FAO flux on the other. To explore the relationship between FAO and insulin sensitivity, we measured the change in intra-hepatic lipids (IHL) by MRS and hepatic insulin sensitivity during a 5-hour hyperinsulinemic euglycemic clamp with co-infusion of either a control glycerol solution versus a 20% intralipid solution, in random order and separated by 4 months. The study cohort included 18 subjects with an inherited defect in FAO (FAOD; including Carnitine Palmitoyltransferase 2, Long chain 3-Hydroxyacyl-CoA Dehydrogenase, Very Long-chain or Medium-chain Acyl-CoA Dehydrogenase Deficiencies) and 18 matched normal healthy controls. Endogenous glucose production (EGP) before the start of the insulin infusion was similar between FAOD and controls. In controls, insulin suppressed EGP during the glycerol control clamp but this effect was significantly reduced during intralipid infusion (P<0.05, reduced hepatic insulin sensitivity) . In FAOD subjects, insulin suppressed EGP similarly during the glycerol and intralipid clamps (no impairment in insulin action) . Untargeted lipidomics /metabolomics of plasma measured at the end of the intralipid clamp demonstrated significant elevations of plasma acylcarnitines and some triglyceride species but lower production of ketones, and several phospholipids in subjects with FAOD compared to controls. Neither increased IHL nor elevated plasma acylcarnitines in FAOD subjects were associated with hepatic insulin resistance. In fact, impaired FAO protected against intralipid-induced hepatic insulin resistance suggesting that increased FAO flux rather than impaired mitochondrial function is a major determinant of insulin resistance in the liver.

Disclosure

J.Q.Purnell: Advisory Panel; Novo Nordisk. A.N.Gregor: None. D.Choi: None. E.Baetscher: None. W.Rooney: Consultant; Ultragenix, Research Support; Italofarmco, Revalesio. C.O.Harding: None. M.B.Gillingham: None.

Funding

NIH/NIDDK: R01DK102813NIH/NCATS: UL1TR002369
228-LB: Mitochondrial Architecture Controls Fatty Acid Utilization by Regulating CPT1 Sensitivity to Malonyl-CoA
Authors: NGO JENNIFER , WOOK CHOI DONG , STILES LINSEY , DIVAKARUNI AJIT S. , LIESA MARC , DANIAL NIKA N. , SHIRIHAI ORIAN
Published: May 2022

Mitochondrial network fragmentation has been associated with metabolic dysregulation, although the underlying mechanisms remain unknown. Herein, using multiple cellular models where the physiological relevance of fatty acid is well-established, we show that mitochondrial fragmentation is highly associated with increased fatty acid oxidation (FAO) rates. Through confocal imaging and stable isotope tracing analysis, forced mitochondrial elongation using Mfn2 over-expression or Drp1 depletion significantly decreased FAO rates, while Mfn2 knockout leads to enhanced FAO rates (P-value =0.0003) , suggesting that mitochondrial morphology regulates fatty acid utilization. To determine the mechanism by which mitochondrial architecture controls fatty acid utilization we studied the effects of mitochondrial fragmentation and elongation on CPT1 activity. Remarkably, we find that CPT1 sensitivity to its main regulator, malonyl-CoA is strongly affected by molecular interventions that inhibit mitochondrial fusion and fission. Notably, the reduction in hepatic FAO rates upon mitochondrial elongation is attributed to a 14 fold increase in CPT1 sensitivity to malonyl-CoA. Consistent with this mechanism we show that the inhibition of FAO imposed by mitochondrial elongation can be overcome using CPT1 pharmacological activator that is malonyl-CoA-independent, but not by the malonyl-CoA-sensitive activator. Furthermore, we tested the functional implications of this mechanism in hepatocytes and beta cells. In hepatocytes we show that forced elongation by the inhibition of fission reduced gluconeogenesis by 30-70%. Whereas in islets, forced mitochondrial fragmentation through molecular inhibition of fusion, resulted in a 2.5 fold increase in insulin secretion at basal glucose concentrations, a phenomenon associated with prediabetes and obesity. Taken together, our study provides a biochemical and mechanistic explanation by which changes to mitochondrial architecture affects fuel utilization and lipid metabolism.

Disclosure

J. Ngo: None. D. Choi: None. L. Stiles: None. A. S. Divakaruni: None. M. Liesa: None. N. N. Danial: None. O. Shirihai: None.

Funding

American Diabetes Association (1-19-IBS-049)
236-LB: Sex Differences in Lipid Storage and Metabolism Underlay Dimorphic Inflammatory Responses in Obesity
Authors: MOHAN RAMKUMAR , VARGHESE MITA , ABRISHAMI SIMIN , SINGER KANAKADURGA
Published: May 2022

Obesity-induced inflammation is a risk factor for metabolic impairment leading to diabetes and cardiovascular disease. We, and others, have shown that sex differences exist in adipose tissue (AT) and myeloid inflammatory responses to high-fat diet (HFD) and lipolysis. However, the causal cellular mechanisms behind this sexual dimorphism are not well defined. Our objective was to investigate sex differences in lipid storage and metabolism that drive sexually dimorphic inflammatory responses. C57Bl/6J mice were fed a normal diet or 60%kcal%fat (HFD) for 16 weeks. AT macrophages (ATMs) were sorted for RNA sequencing. Animals were gonadectomized (GX) prior to starting HFD and gonadal white AT (GWAT) was collected for lipidomics. To directly model androgen receptor (AR) activation, GX mice were given dihydrotestosterone (DHT) to study AR-specific effects. RNA sequencing identified upregulated genes (Lpl, Ctsb, and Cd36) linked to a metabolically-activated macrophage only in male HFD ATMs. Furthermore, pathways associated with mitochondrial depolarization and fatty acid (FA) metabolism differed significantly by sex. GWAT lipidomics identified shifts in lipid species both by sex and hormone status, and differences were also observed in acylcarnitines and saturation of ceramide species that are implicated in inflammation. In addition, males showed elevated levels of key precursor free FAs (palmitic, linoleic, and arachidonic acid) when compared to female and GX mice. DHT treatment did not completely mimic male pro-inflammatory responses but induced GWAT crown-like structures in both sexes. DHT failed to upregulate inflammatory markers (Il6 and Mcp1) but showed impaired expression of adipogenic genes (Pparg and Pgc1α) in GX mice. Altogether, our data suggest there are sex-specific alterations in GWAT lipid storage and metabolism that may drive sexually dimorphic ATM polarization, which is only partially reversed by AR activation.

Disclosure

R. Mohan: None. M. Varghese: None. S. Abrishami: None. K. Singer: None.

Funding

National Institutes of Health - NIDDK (R01DK115583)
236-OR: Sleep Deprivation Prevents Counterregulatory Adaptation to Recurrent Hypoglycaemia
Authors: MEYHOEFER SVENJA , DEMBINSKI KATHARINA , SCHULTES BERND , JAN BORN , WILMS BRITTA , LEHNERT HENDRIK , HALLSCHMID MANFRED , MEYHÖFER SEBASTIAN M.
Published: May 2022

Aims/Hypothesis: Attenuated counterregulation after recurrent hypoglycaemia is a major complication of diabetes treatment. Considering previous evidence for the relevance of sleep in metabolic control, we assessed the acute contribution of sleep to the counterregulatory adaptation to recurrent hypoglycaemia.

Methods: Within a balanced cross-over design, 15 healthy, normal-weight male participants aged 18-35 years underwent three hyperinsulinaemic-hypoglycaemic clamps with a glucose nadir of 2.5 mmol/L, under two experimental conditions, sleep and sleep deprivation (SD) . Participants were exposed to two hypoglycaemic episodes, followed by a third hypoglycaemic clamp after one night of regular 8-hour sleep vs. SD. The counterregulatory response of relevant hormones (glucagon, growth hormone (GH) , adrenocorticotropic hormone (ACTH) , cortisol, epinephrine, and norepinephrine) . Moreover, autonomic and neuroglycopenic symptoms were assessed.

Results: SD compared to sleep dampened the adaptation to recurrent hypoglycaemia of epinephrine (p=0.004) , and this pattern also emerged in an overall analysis including epinephrine, GH, and glucagon (p=0.064) . Whereas after regular sleep, counterregulatory responses of epinephrine (p=0.005) , GH (p=0.029) , and glucagon (p=0.009) were attenuated during the third compared to the first clamp, they were preserved after SD (all p>0.225) . Neuroglycopenic and autonomic symptoms during the third compared to the first clamp were likewise reduced after sleep (p=0.0 and p=0.019, respectively) . In SD, neuroglycopenic symptoms increased (p=0.014) and autonomic symptoms remained unchanged (p=0.859) .

Conclusions/Interpretation: The counterregulatory adaptation to recurrent hypoglycaemia is compromised by SD between hypoglycaemic episodes, indicating that sleep is essential for the formation of a neuro-metabolic memory and a potential target of interventions to improve hypoglycaemia unawareness.

Disclosure

S.Meyhoefer: n/a. K.Dembinski: None. B.Schultes: None. B.Jan: None. B.Wilms: None. H.Lehnert: None. M.Hallschmid: None. S.M.Meyhöfer: None.

Funding

DFG (German Research Funding Organisation) : TR-SFB654-B1
239-LB: Single-Cell Analysis Identified Inhibitory Neurons Associated with High-Fat Diet in the Bed Nucleus of the Stria Terminalis
Authors: YANG XIUXIU , FLAK JONATHAN , JOHNSON TRAVIS S.
Published: May 2022

Obesity occurs due to inadequate homeostatic function controlled by the central nervous system (CNS) . Outside of a few brain areas, little information is available on structures, such as the bed nucleus of the stria terminalis (BST) and preoptic area (POA) , that are essential for body weight regulation. To better understand BST and POA mechanisms involved in obesity, we overlayed deep sequencing results collected from obese (by Echo MRI) and diabetic (by glucose tolerance test) mice on high fat diet (HFD) with a previously published single cell RNA-seq data set. We identified neural subtypes using Seurat (Fig. 1A) and deconvoluted the RNA-seq with CIBERSORT. The Inhibitory 1 subtype had a lower proportion in HFD BST than control BST (Fig. 1B, p=0.035) but no difference was found between BST and POA (Fig. 1C, p=0.505) . We performed differential gene expression analysis to identify Inhibitory 1 markers and associated pathways. Aminobutyric acid (GABA) -ergic synapse pathway was enriched (Fig. 1D,E, p< 0.0001) , which is essential to the CNS control of body weight. HFD induced strong transcriptional changes in BST Inhibitory 1 markers between HFD and control diet (Fig. 1F) . One gene of particular interest was Glutathione Peroxidase 3 (GPX3, p<0.0001) . GPX3, localized to the BST and POA (Fig. 1G) , is down-regulated in both prostate and adipose tissues during HFD and implicated in neurodegenerative disease, but not yet been studied in the brain and obesity. Future studies will investigate BST GPX3 in obesity.

Disclosure

X. Yang: None. J. Flak: Research Support; Eli Lilly and Company. T. S. Johnson: Research Support; Eli Lilly and Company.
248-LB: Nrf2 Is Essential for Expanding Neonatal Pancreatic Beta-Cell Mass
Authors: BAUMEL-ALTERZON SHARON , GARCIA-OCANA ADOLFO , SCOTT DONALD
Published: May 2022

Patients with both major forms of diabetes suffer from insufficient functional β-cell mass. Moreover, the sharp increase in the annual incidence of both type 1 (1.8%) and type 2 (4.8%) diabetes in the pediatric population in the last decades highlights the urgent need for discovering the mechanisms that regulate functional β-cell mass. β-cell proliferation, which reaches its maximal peak during postnatal stages is the major mechanism for increasing β-cell mass in early postnatal ages. We previously found that induction of the antioxidant regulator, Nrf2, is required for adaptive β-cell expansion under hypercaloric conditions via stimulation of β-cell proliferation, increased β-cell survival, and maintenance of β-cell identity in adult mice. Based on these observations, we hypothesize that Nrf2 regulates β-cell proliferation and survival at early stages of life and contribute to maintain normal β-cell mass later in life. Nrf2 expression levels were found to correlate with β-cell proliferation in wild type neonatal mice. Importantly, 28 day-old β-cell specific Nrf2 knockout mice (Ins1Cre-Nrf2lox/lox) exhibited 50% reduction in β-cell mass, 58% reduction in total islet number, 43% decrease in extra-large islets, 46% decrease in large islets and 53% decrease in extra small islets, compared to control mice. No changes in pancreas weight were observed. Additionally, depletion of Nrf2 in β-cells increased β-cell death in 7 (5.8-fold) and 14 (12.5-fold) day-old mice and attenuated β-cell proliferation in 1- and 7-day-old mice. Surprisingly, we also found a marked decrease in islet size in 1- and 7-day-old mice, suggesting that Nrf2 may affect β-cell proliferation, survival and/or development during embryonic stages. We conclude that Nrf2 is required for β-cell mass expansion in postnatal ages by controlling β-cell proliferation and survival.

Disclosure

S. Baumel-alterzon: None. A. Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. D. Scott: None.
260-LB: Elucidating the Role of NTPDase3 and Purinergic Signaling in Islet Hormone Secretion
Authors: RICHARDSON TIFFANY M. , DAI CHUNHUA , ARAMANDLA RADHIKA , DURAI HEATHER H. , REIHSMANN CONRAD , DAVIS COREY , MEI SHAOJUN , GIBSON DANNIELLE , SHRESTHA SHRISTI , SAUNDERS DIANE C. , COATE KATIE C. , ROBSON SIMON C. , POWERS ALVIN C. , BRISSOVA MARCELA
Published: May 2022

ATP release from β cell secretory vesicles during insulin exocytosis is postulated to modulate islet hormone secretion via purinergic signaling in an autocrine or paracrine manner. Ecto-enzymes, including a family of ectonucleotidases (NTPDase) , control the concentration of extracellular nucleotides. One member of this family, ectonucleotidase triphosphate diphosphohydrolase 3 (NTPDase3) , is specifically expressed in human β cells during postnatal maturation, and its enzymatic activity is present in isolated adult human islets. The role of NTPDase3 in islet hormone secretion is not understood. We hypothesized that NTPDase3 regulates islet hormone secretion by modulating local extracellular nucleotide levels that signal through purinergic receptors expressed by islet cells. To define the cellular distribution of purinergic ecto-enzymes and nucleotide receptors in human islets, we used single-cell RNA sequencing (scRNA-seq) data. In addition to β cell NTPDase3 expression, ecto-enzymes ENPP1, and ENPP2 were abundant in α and ε cells while ecto-5'-NT and ADA were expressed in endothelial and stellate cells. These ecto-enzymes can further hydrolyze β cell-derived ATP to ADP, AMP, and adenosine which all bind purinergic receptors. We found that the ATP receptor, P2RY1, was enriched in β cells, and an adenosine receptor, ADORA2A, was expressed in α cells by both bulk and scRNA-seq. To study the function of this purinergic niche, we further assessed extracellular ATP levels which increased concomitantly (4.2E-12 vs. 4.3E-13 moles/100 IEQ/hour; n=9; p=0.03) with insulin secretion (21 vs. 60 ng/100 IEQ/hour; n=9; p=0.02) upon stimulation with 16.7 mM glucose + IBMX. To directly test NTPDase3 loss of function, we genetically manipulated human pseudoislets using an shRNA knockdown approach, resulting in a 1.7-fold increase of insulin secretion (AUC=3 vs. 5 ng/100 IEQs; n=3) . Overall, these studies provide an insight into how extracellular nucleotides and purinergic signaling mechanisms fine-tune human islet hormone secretion.

Disclosure

T. M. Richardson: None. D. C. Saunders: None. K. C. Coate: None. S. C. Robson: Advisory Panel; eGenesis, Consultant; Puretech, Synlogic, Research Support; Tizona Inc, Stock/Shareholder; EPurines, Purinomia. A. C. Powers: None. M. Brissova: None. C. Dai: None. R. Aramandla: None. H. H. Durai: None. C. Reihsmann: None. C. Davis: None. S. Mei: None. D. Gibson: None. S. Shrestha: None.
285-OR: Economic Impact of Novel Interventions in Children’s Health Care (NICH) in Youth with T1D
Authors: WAGNER DAVID V. , MIJANOVICH TOD , BARRY-MENKHAUS SAMANTHA A. , HARRIS MICHAEL A.
Published: May 2022

Objective: Youth with T1D and high social risk experience health disparities, including acute complications. Novel Interventions in Children’s Healthcare (NICH) , designed to target social risk, is associated with improved health, fewer complications, and lower medical costs. This is the first study to compare NICH outcomes in youth with T1D to a control group using claims data.

Methods: Using a state-based claims database, a difference-in-differences study with a matched comparison group was conducted, comparing youth with T1D served by NICH (n=25) to youth referred to NICH but denied access by insurance or with similar claims history and demographic characteristics (n=47) . Data across a 36-month follow up period were examined.

Results: Mahalanobis matching and entropy balance weighting created well-balanced groups on matched covariates. NICH reduced hospitalizations by 1.6 (p<.01) , ED visits by 4.1 (p<.01) , and cost by >$18k per youth (p<.05) , with maximum impact in the 2nd follow up year (>$9k, p<.01) .

Conclusion: Youth served by NICH had fewer acute medical events and lower costs following NICH initiation than control youth. Youth with complication history did not “regress to the mean,” and continued to experience acute health emergencies - highlighting the potential value of interventions that target youth with T1D and high social risk in need of alternatives to standard medical and behavioral health care.

Disclosure

D.V.Wagner: None. T.Mijanovich: None. S.A.Barry-menkhaus: None. M.A.Harris: None.

Funding

The Leona M. and Harry B. Helmsley Charitable Trust
294-OR: The Type 1 Diabetes Protective Variant of Tyrosine Kinase 2 (TYK2) Protects ß-Cells from Autoimmunity
Authors: TAMAYO AYMEE RODRIGUEZ , HUBER MOLLIE , BUTFILOSKI EDWARD J. , MATHEWS CLAYTON E.
Published: May 2022

Type 1 diabetes (T1D) is a complex autoimmune disease where genes and environmental factors impact pathogenesis. Analysis of the >150 loci identified by GWAS reveals the compelling theme that candidate genes cluster into pathways. A pathway connecting environment to T1D genetics is the anti-viral/Type 1 interferon (IFN1) pathway that is regulated by at least 7 T1D linked loci, including Tyrosine Kinase 2 (TYK2) which regulates IFN1 receptor signaling. We test the hypothesis that the T1D protective variant of TYK2 restrains signaling though the IFN1 receptor functions in β-cells reduce interactions with and killing by autoreactive, cytolytic CD8+ T lymphocytes (CTL) . These studies employed β-cells edited in the endogenous TYK2 locus with CRISPR/Cas9 to express the T1D protective TYK2110A or common allotype, TYK2110P. These cells were treated with Poly I:C (50ng/mL) , to see IFN1 response to an imitated viral infection. Outcomes were IFN1 production, gene expression, antigen presentation, and responses to CTL. When allotypic β-cells were treated with Poly I:C, both produced equivalent levels of IFN1. While β-cells with TYK2110P showed increased mechanisms of antigen presentation, this pathway was attenuated in TYK2110A β-cells (p<0.05) . These results were replicated by treating TYK2110P and TYK2110A IFN1 (100U/mL) , where we observed increased antigen presentation in TYK2110P β-cells but not TYK2110A β-cells (p<0.05) . Interestingly, prior to treatment cell surface human leukocyte antigen class I levels were reduced in TYK2110A β-cells (p<0.01) . Studies to test the interactions of autoreactive CTL with β-cells supported our hypothesis. TYK2110P β-cells were efficiently lysed by CTL and lysis was increased by pre-treatment of β-cells with IFN1 or PolyI:C. In stark contrast, TYK2110A β-cells exhibited low levels of CTL-lysis and pre-treatment with IFN1 or PolyI:C did not modify lysis.

In conclusion, TYK2110A protects β-cells from autoimmune-mediated death.

Disclosure

A.Rodriguez tamayo: None. M.Huber: None. E.J.Butfiloski: None. C.E.Mathews: None.

Funding

National Institutes of Health PAI42288National Institutes of Health RR01DK127497
295-OR: Identification of Type 1 Diabetes Genes and Regulatory Processes Mediating Pancreatic Beta-Cell Survival in Response to Proinflammatory Cytokines
Authors: BENAGLIO PAOLA , ZHU HAN , OKINO MEI-LIN , YAN JIAN , ELGAMAL RUTH , NARIAI NAOKI , BEEBE ELISHA , KORGAONKAR KATHA , QIU YUNJIANG , DONOVAN MARGARET , CHIOU JOSHUA , NEWSOME ASHMUS JACKLYN M. , KAUR JASPREET , CORBAN SIERRA , REN BING , FRAZER KELLY , SANDER MAIKE , GAULTON KYLE J.
Published: May 2022

The response of beta cells to inflammatory cytokines contributes to type 1 diabetes (T1D) risk, but the specific genes that underlie this response and mediate beta cell survival remain largely unknown. In this study we used a suite of functional genomics assays combined with human genetics, to map cis-regulatory programs in beta cells and to identify T1D genes that affect beta cell survival upon exposure to the pro-inflammatory cytokines IL1β, IFNγ and TNFα. We mapped 38,931 cytokine-responsive candidate cis­­ -regulatory elements (cCREs) active in beta cells using ATAC-seq and single nuclear ATAC-seq (snATAC-seq) , and linked cytokine-responsive beta cell cCREs to putative target genes using single cell co-accessibility and HiChIP. We performed a genome-wide pooled CRISPR loss-of-function screen in EndoC-βH1 cells, which identified 867 genes affecting cytokine-induced beta cell loss. Genes that promoted beta cell survival and had up-regulated expression in cytokine exposure were specifically enriched at T1D loci, and these genes were preferentially involved in inhibiting inflammatory response, ubiquitin-mediated proteolysis, mitophagy and autophagy. We identified 2,229 variants in cytokine-responsive beta cell cCREs altering transcription factor (TF) binding using high-throughput SNP-SELEX, and variants altering binding of TF families regulating stress, inflammation and apoptosis were broadly enriched for T1D association. Finally, through integration with genetic fine mapping, we identified T1D risk variants regulating beta cell survival in cytokine exposure. At the 16p13 locus, a T1D variant affected TF binding in a cytokine-induced beta cell cCRE that physically interacted with the SOCS1 promoter, and increased SOCS1 activity promoted beta cell survival in cytokine exposure. Together our findings reveal processes and genes acting in beta cells during cytokine exposure that intrinsically modulate risk of T1D.

Disclosure

P.Benaglio: None. M.Donovan: None. J.Chiou: Employee; Pfizer Inc. J.M.Newsome ashmus: None. J.Kaur: None. S.Corban: None. B.Ren: Stock/Shareholder; Arima Genomics, Epigenome Technologies. K.Frazer: n/a. M.Sander: None. K.J.Gaulton: Consultant; Genentech, Inc., Stock/Shareholder; Neurocrine Biosciences, Inc., Vertex Pharmaceuticals Incorporated. H.Zhu: None. M.Okino: None. J.Yan: None. R.Elgamal: None. N.Nariai: None. E.Beebe: None. K.Korgaonkar: None. Y.Qiu: None.

Funding

NIH DK122607
303-OR: Localized CTLA-4-Ig and Antioxidant Islet Encapsulation Delays Allograft Rejection
Authors: BARRA JESSIE , BURNETTE KALIA S. , KOZLOVSKAYA VERONIKA , KHARLAMPIEVA EUGENIA , TSE HUBERT M.
Published: May 2022

Type 1 diabetes is characterized by beta-cell-specific autoimmune destruction, leading to life-long exogenous insulin-dependence. Islet transplantation can restore proper glycemic control, but graft longevity remains low due to immune-mediated rejection. The use of nanothin encapsulation materials consisting of poly (N-vinylpyrrolidone) (PVPON) and tannic acid (TA) , an antioxidant, can delay islet allograft rejection and decrease inflammatory immune responses. However, when (PVPON/TA) -encapsulated C57BL/6 islets were transplanted into streptozocin-treated diabetic NOD mice in the absence of systemic immunosuppression, only 50% of recipients maintained long-term allograft function. Therefore, modification of (PVPON/TA) coatings to include immune inhibitors are needed to prolong islet allograft survival. The adaptability of our (PVPON/TA) layers allow for its conjugation to CTLA-4-Ig, a fusion protein that can block CD28-mediated co-stimulation of T cells. We hypothesize that the addition of CTLA-4-Ig to (PVPON/TA) outer layers, denoted as (PVPON/TA/CTLA-4-Ig) , will extend islet allograft survival by inhibiting proinflammatory effector T cell responses. Transplantation of (PVPON/TA/CTLA-4-Ig) -encapsulated NOD.Rag islets significantly delayed islet allograft rejection in diabetic C57BL/6 mice compared to non- (p<0.0001) and control IgG-containing (PVPON/TA/IgG) -encapsulated islets (p<0.01) . Additionally, (PVPON/TA/CTLA-4-Ig) -encapsulation significantly increased FOXP3+ regulatory and CD73+ FR4+ anergic CD4+ T cell count when compared to both non- and (PVPON/TA/IgG) -encapsulation. Since CTLA-4-Ig can affect the tryptophan catabolism pathway, future studies will investigate the effect of (PVPON/TA/CTLA-4-Ig) -encapsulation on indolamine-2,3-dioxygenase expression in antigen-presenting cells. The success of (PVPON/TA/CTLA-4-Ig) coatings to delay allograft rejection warrant their use in future human translational studies.

Disclosure

J.Barra: None. K.S.Burnette: None. V.Kozlovskaya: None. E.Kharlampieva: None. H.M.Tse: None.

Funding

NIH/NIDDK Raward (DK099550) NIH/NIDDK Raward (DK127497) JDRF award (SRA-2016-270-S-B) JDRF award (2-SRA-2019-692-S-B) NIH/NIDDK R56 award (DK126456) NIH/NIDDK Raward (DK126456) NIH/NIGMS (T32GM109780) NIH/NIGMS (T32GM008111)
31-LB: Improvements in Glycemic Control following Use of an Established mHealth Program among Individuals with Diabetes
Authors: ZIMMERMANN GRETCHEN , VENKATESAN AARATHI , RAWLINGS KELLY , FRANK RICHARD S. , EDWARDS CAITLYN
Published: May 2022

Background: The prevalence of diabetes continues to rise worldwide. mHealth programs with 24/7 app access and frequent interaction with remote providers ease the learning, practice, application, and daily integration of self-care skills and health improvement behaviors for people diagnosed with diabetes, especially those at high risk for cardiometabolic and mental health complications. This study aimed to evaluate the effectiveness of an established mHealth program on measures of glycemic control in a population at high risk for the development of diabetes-related complications (hemoglobin A1c (HbA1c) ≥ 8.0%) .

Methods: This study protocol employed a single-arm, retrospective design. Participants were offered the app-based Vida Health Diabetes Management program as part of their medical insurance benefit. This intervention utilized one-to-one remote sessions with Registered Dietitians and Certified Diabetes Care and Education Specialists as well as structured lessons and tools related to diabetes management. Participants were included if they had a valid laboratory HbA1c completed between 365 to 21-days of enrollment and a follow-up laboratory HbA1c value within 90 to 365-days following enrollment. Two-tailed paired t-tests were used to evaluate changes in HbA1c between baseline and follow-up.

Results: Participants with HbA1c ≥ 8.0% at baseline (n=1023) demonstrated a decrease in HbA1c of -1.37 points between baseline (mean: 9.84, SD: 1.64) and follow-up (mean: 8.47, SD: 1.77, p<0.001) . Additionally, we observed a decrease in HbA1c of -1.94 points between baseline (mean: 10.77, SD: 1.48) and follow-up (mean: 8.83, SD: 1.94, p<0.001) among participants with HbA1c ≥ 9.0% (n = 618) .

Conclusion: This study demonstrated clinically meaningful improvements in glycemic control among participants enrolled in the Vida Health Digital Diabetes Management Program. Future work should aim to identify mechanisms of this, and other mHealth interventions, most associated with glycemic control.

Disclosure

G. Zimmermann: Employee; Vida Health. A. Venkatesan: Employee; Vida Health. K. Rawlings: Employee; Vida Health. R. S. Frank: Employee; Vida Health. C. Edwards: Employee; Vida Health.

Funding

Vida Health
312-OR: Glucose Oxidation in Brown Fat
Authors: JIANG LEI , WANG QIONG (ANNABEL) , WANG ZHICHAO
Published: May 2022

In the United States, more than 70% of adults are overweight or obese, caused primarily by excess energy intake in modern society. Brown adipose tissue (BAT) can function as an “energy sink” in mammals, since BAT is specialized with uncoupling respiration, which burns energy to maintain body temperature. Unfortunately, thermogenesis in brown adipocytes dramatically declines in obese individuals, contributing to the development of obesity-related metabolic disorders. Although both lipids and glucose have been viewed as major energy substrates for thermogenesis, most of the early studies have been focusing on fatty acid oxidation. Recently, we found that BAT had the most active glucose oxidation among metabolic active tissues, and chronic cold exposure significantly enhanced glucose oxidation in mice BAT. Furthermore, in vivo administration of a mitochondrial pyruvate carrier (MPC) inhibitor impaired glucose oxidation and thermogenesis in BAT, which was consistent with the impaired thermogenesis of a recently published mouse model of constitutive BAT-selective deletion of MPC1 (Ucp1-MPC1-KO) . In order to directly study the role of MPC in mature brown adipocytes, we generated a Tet-on inducible Ucp1‑specific MPC2 knockout (Ucp1‑MPC2‑iKO) mouse model. As expected, Ucp1-MPC2-iKO mice couldn’t maintain body temperature upon acute cold exposure, after prolonged loss of MPC in mature brown adipocytes. Interesting, adult Ucp1-MPC2-iKO mice were cold-resistant, after the acute loss of MPC in the mature brown adipocytes, which were different from the constitutive Ucp1-MPC1-KO mice. Together, our study suggests that although glucose oxidation is essential to optimal BAT thermogenesis, MPC-independent metabolism exists to maintain glucose oxidation and thermogenesis in BAT.

Disclosure

L. Jiang: None. Q. Wang: None.
314-OR: Synaptotagmin-9 Regulates Early Phase Insulin Secretion by Regulating Tomosyn-1
Authors: RAHMAN MD MOSTAFIZUR , ALSHARIF HAIFA , BHATNAGAR SUSHANT
Published: May 2022

Insulin secretion from pancreatic β-cells occurs in a biphasic manner. The loss of insulin secretion, entailing the inability of insulin granules to fuse with the plasma membrane (PM) via the formation of the SNARE complex, is a key determinant in impaired glucose tolerance and development of type 2 diabetes (T2D) . Synaptotagmins (Syt) are calcium sensors that regulate insulin-containing granule fusion to the PM for insulin secretion. Syt9 is highly expressed in β-cells; however, its role in insulin secretion is not well-characterized. Our goal is to determine how Syt9 affects insulin secretion from pancreatic β-cells. Mice lacking Syt9 (Syt9-/-) have improved glucose clearance with no change in insulin action than wild-type control mice. Interestingly, Syt9-/- mice showed increased plasma insulin levels at 5 min and 15 min post-glucose challenge. Similarly, islets isolated from Syt9-/- mice demonstrate increased stimulation of insulin secretion in response to the early phase insulin secretagogues (KCl and tolbutamide) compared to WT control islets. Similarly, siRNA-mediated knockdown of Syt9 in INS1 cells increased KCL-stimulated insulin secretion at 5 min and min. Altogether, these data suggest that Syt9 regulates the early phase of insulin secretion from β-cells. Strikingly, Syt9-/- mouse islets have reduced Tomosyn-1 levels (∼50%) without altering the levels of other key SNARE proteins (Stx1A, Stx4A, SNAP25) . Tomosyn-1, an inhibitor of insulin secretion whose increased protein abundance is associated with reduced insulin secretion, binds, cofractionates, and colocalizes with Syt9 and Stx1A. The knockdown of Tomosyn-1 in INS1 β-cells treated with KCL prevents the localization of Syt9 with Stx1A. Further, the knockdown of Syt9 or Tomosyn-1 increases the SNARE complex formation. In sum, these data demonstrate the identification of a novel Stx1A-Tomosyn-1-Syt9 molecular complex that regulates the fusion of insulin granules in the early phase.

Disclosure

M.Rahman: None. H.Alsharif: n/a. S.Bhatnagar: None.

Funding

R01DK120684-01
321-OR: Empagliflozin Improves Beta-Cell Function Independently of Glucose Toxicity in Patients with Type 2 Diabetes
Authors: THIRUMATHYAM ROOPAMEERA , RICHTER ERIK A. , GOETZE JENS PETER , MADSEN PER L. , HOLST JENS J. , MADSBAD STEN , JØRGENSEN NILS B.
Published: May 2022

Introduction: SGLT2-inhibitors improve beta-cell glucose sensitivity (BGS) and insulin sensitivity in patients with type 2 diabetes (T2D) . This effect has been attributed to relief of glucotoxicity, but very few studies have controlled for differences in glycemia. Here, we explore the effects of empagliflozin on BGS and secondarily, peripheral insulin sensitivity (IS) , compared to similar glycemic control with insulin treatment.

Materials and Methods: 17 subjects with T2D (13 males, 58±3 years (mean±sem) , BMI 32.9±0.9 kg/m2, HbA1c 52.4±2.4 mmol/L, T2D duration 8.9±1.3 years) were treated with empagliflozin (E) and NPH-insulin (I) for 5 weeks in a randomized cross-over study design with 3 weeks of washout. Insulin was titrated to obtain similar glycemic control as with empagliflozin. An OGTT with stable iv and oral glucose tracers was performed prior to and at the end of each treatment.

Results: Glycemia was similarly reduced on both treatments (Δfasting glucose: E: -1.3±0.2, p<0.01; I: -0.8±0.2, p<0.01; E vs. I: p=NS) . Fasting insulin concentrations were lower with empagliflozin and higher with insulin (E: 92±12 vs. 180±27 pmol/L, p<0.001) . Fasting hepatic glucose production (HGP) was increased with empagliflozin compared to insulin (19.5±0.8 vs. 16.6±0.6 µmol/kg/min, p<0.001) , but postprandial HGP did not differ between treatments. BGS was greater with empagliflozin compared to insulin (0.82±0.1 vs. 0.59±0.pmol/ (kg·min·mM) , p=0.001) . IS (AUC Glucose Rd, tissue /AUC Insulin) was unchanged with empagliflozin, but decreased with insulin compared to washout (0.09±0.vs. 0.11±0.02 µmol/ (kg FFM·pM·min) , p=0.025) . Fatty tissue insulin resistance (Adipo-IR: fasting FFA x fasting insulin) was lower with empagliflozin compared to insulin (77±vs. 110±17 mmol/L x pmol/L, p=0.012) .

Conclusion: Improved beta-cell function during empagliflozin treatment is not a result of reduced glucotoxicity. Peripheral hyperinsulinemia may induce insulin resistance.

Disclosure

R.Thirumathyam: None. E.A.Richter: None. J.Goetze: Consultant; Novo Nordisk A/S. P.L.Madsen: None. J.J.Holst: Advisory Panel; Novo Nordisk, Board Member; Antag Therapeutics, Bainan Biotech. S.Madsbad: None. N.B.Jørgensen: Advisory Panel; Novo Nordisk A/S, Research Support; Boehringer Ingelheim International GmbH, Speaker's Bureau; Novo Nordisk A/S, Stock/Shareholder; Eli Lilly and Company, Novo Nordisk A/S.

Funding

Boehringer Ingelheim Gmbh Investigator initiated study grant