bluuebunny/crossref_metadata_2025_split huggingface dataset:
208-OR: Which Diabetes Strengths Matter Most? Associations with Key Outcomes
Authors: CAO VIENA T. , ESHTEHARDI SAHAR S. , THOMPSON DEBORAH , KARAVITI LEFKOTHEA , HILLIARD MARISA E. 
Published: June 2019

Objectives: Many adolescents use adaptive type 1 diabetes (T1D)-related behaviors, or “strengths,” to overcome challenges and achieve resilient diabetes outcomes (i.e., good self-management, general and T1D-specific quality of life (QoL), and glycemic control). Overall, strengths are linked to these key outcomes, but identifying which individual positive behaviors are most associated may assist care teams in promoting the most important strengths in their patients.
Methods: Participants were n=82 adolescents (age 12-17, M age=15.3±1.5 years, 59% female, M HbA1C=8.9±2.0%, 55% pump) with T1D enrolled in a behavioral intervention. Baseline data included youth self-report on the Diabetes Strengths and Resilience (DSTAR) measure (12 items, 2 scales) and measures of self-management and QoL, and chart-confirmed HbA1c.
Results: Significant correlations between DSTAR items/scales and diabetes outcomes are summarized in the Table.
Conclusions: T1D strengths are related to all key outcomes. The items about overall T1D-related self-efficacy and prioritizing T1D management are related to multiple resilient outcomes and may be important intervention targets. Both DSTAR scales and 9 of their items correlated with self-management, suggesting strengths-based interventions may benefit treatment adherence. Youth with better outcomes may also feel more capable. Unexpected inverse associations for 3 items should be further studied.

Disclosure
V.T. Cao: None. S.S. Eshtehardi: None. D. Thompson: None. L. Karaviti: None. M.E. Hilliard: None.

Funding
National Institute of Diabetes and Digestive and Kidney Diseases (1R21DK107951)
2082-P: Pharmacological Induction of Brown and Beige Adipose Tissue
Authors: FINLIN BRIAN , MEMETIMIN HASIYET , CONFIDES AMY L. , ZHU BEIBEI , JOHNSON ZACHARY R. , DUPONT-VERSTEEGDEN ESTHER E. , KERN PHILIP A. 
Published: June 2019

Brown adipose tissue (BAT) is associated with improved metabolic homeostasis in humans, and studies in rodents indicate that subcutaneous white adipose tissue (SC WAT) beiging, which involves increased uncoupling protein 1 (UCP1) expression, also is associated with improved metabolism. The goal of this study was to determine the ability of mirabegron (a β3AR agonist), pioglitazone (a thiazolidinedione), or a combination of the drugs to induce brown and beige adipose tissue and to determine the effects on glucose and lipid homeostasis. We randomized obese, insulin-resistant (IR) research participants to mirabegron (50 mg/day), pioglitazone (30 mg/day), or combination therapy treatment groups. Euglycemic clamping, oral glucose tolerance tests, adipose tissue biopsies, and PET-CT scans were performed at baseline and after 10 weeks of treatment. Mirabegron improved glucose homeostasis (reduced HbA1c and improved oral glucose tolerance) to a similar extent as pioglitazone without side effects or weight gain. Although mirabegron treatment increased insulin sensitivity, the effect size was much smaller than pioglitazone, yet mirabegron treatment significantly increased the insulinogenic and disposition indexes, suggesting that a major part of the mechanism of mirabegron action involved improving β-cell function. Mirabegron treatment consistently induced SC WAT beiging as evidenced by increased UCP1 expression (2.4 fold increase; P&lt;0.0001), but did not induce BAT, suggesting that induction of beige adipose may be part of the mechanism responsible for improved glucose homeostasis. Pioglitazone also induced beiging (1.6 fold increase in UCP1; P&lt;0.01), but not BAT. The combination of mirabegron plus pioglitazone was not additive, with similar effects on glucose tolerance as single drug. These results suggest that mirabegron treatment has significant beneficial metabolic effects in obese, IR subjects through a distinct mechanism involving SC WAT beiging and improved pancreatic beta cell function.

Disclosure
B. Finlin: None. H. Memetimin: None. A.L. Confides: None. B. Zhu: None. Z.R. Johnson: None. E.E. Dupont-Versteegden: None. P.A. Kern: None.

Funding
National Institutes of Health (R01DK112282)
2086-P: The Interaction of Fitness, Body Mass Index, and Mortality Risk in Type 2 Diabetes
Authors: NYLEN ERIC , KOKKINOS PETER , FASELIS CHARLES , NARAYAN PUNEET , KARASIK PAMELA , MYERS JONATHAN N. 
Published: June 2019

Introduction: Obesity and poor cardiorespiratory fitness (CRF) commonly coexists in type 2 diabetes (T2DM). Moreover, CRF has been associated with lower mortality risk in T2DM patients. In this study, we assessed the modulatory impact of fitness on mortality risk in T2DM patients across the obesity spectrum.
Methods: We identified 6,888 T2DM patients (age 58.3+10.1 years) with no evidence of ischemia as indicated by a maximal standardized exercise treadmill test. We established two CRF categories based on age-adjusted median peak metabolic equivalents (METs) achieved: Unfit (5.4±1.3 METs; n=3,474) and Fit (9.0±2.3 METs; n=3414). We also defined the cohort based on body mass index (BMI) normal-weight (BMI 18.5-24.9 kg/m2); over-weight (BMI 25.0-29.9 kg/m2); and obese (BMI ≥30 kg/m2). We then combined the BMI and CRF categories to form a total of six groups CRF-BMI categories. Cox proportional hazards models, adjusted for known risk factors were constructed to assess the association between CRF-BMI interaction and mortality risk.
Results: During a follow-up of 14.2+6.9 years, 2,495 patients died, accounting for 25.5 events/1,000 person-years of observation. When CRF-BMI groups were considered, the normal-weight-Fit category was used as the referent. Mortality risk in the normal-weight but Unfit patients was 64% higher compared to their Fit counterparts (HR 1.64; 95% CI: 1.35-2.00). In the overweight category, mortality risk was 20% lower in the Fit (HR 0.80; 95% CI: 0.67-0.94) and 28% higher (HR 1.28; 95% CI: 1.09-1.51) in Unfit patients. In obese category the risk was 29% lower for the Fit (HR 0.71; 95% CI: 0.60-0.86) and 23% higher for the Unfit patients (HR 1.23; 95% CI: 1.05-1.45).
Conclusion: Increased CRF favorably modulates mortality risk across the obesity spectrum in T2DM patients. Poor fitness had a substantially greater and negative impact on Normal than over-weight and obese individuals.

Disclosure
E. Nylen: None. P. Kokkinos: None. C. Faselis: None. P. Narayan: Research Support; Self; AstraZeneca, Eli Lilly and Company. P. Karasik: None. J.N. Myers: None.
210-OR: A Combined Method Improves Risk Prediction for Childhood Type 1 Diabetes in the TEDDY Study
Authors: FERRAT LAURIC A. , VEHIK KENDRA , SHARP SETH A. , LERNMARK ÅKE , ZIEGLER ANETTE , REWERS MARIAN , SHE JIN-XIONG , TOPPARI JORMA , AKOLKAR BEENA , KRISCHER JEFFREY , WEEDON MICHAEL N. , RICH STEPHEN S. , ORAM RICHARD A. , HAGOPIAN WILLIAM ,   TEDDY STUDY GROUP
Published: June 2019

Background: There is an unmet need for accurate cost-effective estimation of future T1D risk.
Methods: We derived a combined T1D prediction model using 7883 children followed closely from birth for a median of 9 yr, considering T1D genetic risk score (GRS), family T1D history (FH), standard islet autoantibodies (IA), race, birth circumstances, early growth and nutrient status. T1D developed in 326.
Results: Machine learning and traditional methods (Cox models) performed equivalently as measured by time-dependent AUC ROC. Future T1D was accurately predicted by combining only 3 variables: GRS, FH and IA status. Accuracy of combined scores increased with age at scoring. By age 2, they were highly predictive for T1D in the next 5 years (AUC &gt;0.91, 95% CI 0.88-0.95) (see X on Figure). A 2-yr-old with 2 IA, positive FH and high GRS (&gt;12) would have T1D risk over the next 1, 3 and 5 years of 14% (8-19%), 36% (25-45%) and 51% (39%-61%) respectively. A 2-yr-old with 1 IA, no FH and moderate GRS (10-11) has T1D risk of 0.8% (0.6-1.2%), 2.6% (1.9-3.2%) and 4.3% (3.4%-5.2%) respectively. After newborn genetic screening, only simple venous sampling in routine healthcare settings is required.
Conclusion: This approach allows updated individual risk estimates by age, and in the future may enable release of low risk individuals from surveillance long after initial newborn screening for more cost-efficient population based pediatric T1D prediction.

Disclosure
L.A. Ferrat: None. K. Vehik: None. S.A. Sharp: None. Å. Lernmark: None. A. Ziegler: None. M. Rewers: None. J. She: None. J. Toppari: None. B. Akolkar: None. J. Krischer: None. M.N. Weedon: None. S.S. Rich: None. R.A. Oram: Other Relationship; Self; Randox Laboratories Ltd. W. Hagopian: Research Support; Self; Novo Nordisk A/S.

Funding
National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of Allergy and Infectious Diseases; Eunice Kennedy Shriver National Institute of Child Health and Human Development; National Institute of Environmental Health Sciences; JDRF; Centers for Disease Control and Prevention
2106-P: Subcutaneous Abdominal Fat Has a Stronger Association with Prediabetes than Visceral Fat in Young Indians
Authors: WAGH RUCHA H. , BAWDEKAR RUPALI U. , ALENAINI WAREED , THOMAS ELIZABETH L. , BELL JIMMY DAVID , YAJNIK CHITTARANJAN S. 
Published: June 2019

Background: India is the world’s capital of ‘thin-fat’ phenotype and young onset diabetes. Abdominal adiposity is a possible explanation. We studied the association of abdominal fat distribution by MRI with glucose-insulin metabolism.
Methods: Young adults [18 years, n=595 (308 boys), PMNS cohort] were classified by ADA 75 g OGTT as NGT and prediabetic. We measured anthropometry, total body fat (DXA), and glucose-insulin parameters (HOMA, Insulinogenic and Matsuda index), and visceral (VAT) and subcutaneous (SAT) abdominal adiposity by MRI.
Results: Prediabetic boys were shorter, had a higher BMI, WHR, VAT and SAT compared to NGT boys. Conversely, prediabetic girls had similar BMI, VAT and SAT. SAT was twice as strongly associated than VAT with glucose, insulin resistance, triglycerides and cholesterol concentrations. On multiple regression SAT remained strongly and independently associated.
Conclusion: Unlike in Europeans, SAT has a stronger ‘detrimental’ association with cardio-metabolic risk factors compared to VAT. This may indicate a different adipose biology in Indians and needs further investigation.

Disclosure
R.H. Wagh: None. R.U. Bawdekar: None. W. Alenaini: None. E.L. Thomas: None. J.D. Bell: None. C.S. Yajnik: None.
2110-P: Increased Proinflammatory Eosinophils in Adipose Tissue of Metabolic Syndrome
Authors: MOUSSA KARINE , GURUNG PURNIMA , DEVARAJ SRIDEVI , JIALAL ISHWARLAL 
Published: June 2019

Metabolic Syndrome (MetS) is a common disorder that predisposes to type 2 diabetes mellitus (T2DM) and cardiovascular disease (ASCVD). Adipose tissue (AT) contributes significantly to increased inflammation and insulin resistance (IR) in MetS. Many studies illustrate the role of macrophages and lymphocytes in human SAT, but there is sparse data on other immune cells, especially eosinophils. Hence, in this report we investigated the abundance of eosinophils (EOS) in subcutaneous AT (SAT) of 20 patients with MetS without diabetes, ASCVD, smoking or any inflammatory condition, and matched controls. SAT EOS were quantified by immunochemistry using the Vital New Red stain (ABCAM). Both circulating and SAT EOS were significantly increased 2-fold in MetS (p&lt;0.007) and correlated with each other (r=0.40, p=0.03). Circulating EOS correlated significantly (p&lt;0.05) with triglycerides (TG) (r=0.56), HDL-cholesterol (r=-0.45), IL-6 (r=0.57) and chemerin (r=0.61). SAT EOS correlated significantly (p&lt;0.05) with cardio-metabolic factors including glucose (r=0.42), TG (r=0.39), and adipose IR(r=0.64), biomarkers of inflammation, including leptin (r=0.41), IL-6 (r=0.44), adiponectin (r=-0.39), endotoxin (r=0.56), and chemerin, (r=0.57), and SAT markers of fibrosis (collagen (r=0.42) and Sirius red (r=0.44)).
In conclusion, we make the novel observation that eosinophils are increased in MetS and that SAT EOS are pro-inflammatory and promote fibrosis in SAT. Hence, they contribute to the dysregulation of SAT biology in MetS.

Disclosure
K. Moussa: None. P. Gurung: None. S. Devaraj: None. I. Jialal: None.
2120-P: Knockdown of ETV5 Enhances Nitric Oxide (NO) Production and Caspase-Independent Cell Death in Pancreatic Beta Cells
Authors: LIU FUPENG , PÄTH GÜNTER , LAUBNER KATHARINA , SEUFERT JOCHEN 
Published: June 2019

ETV5 (E-twenty six variant gene 5) belongs to the E-twenty six transcription factor family that regulates various biological processes including proliferation, differentiation and apoptosis. Genome-wide association studies linked ETV5 with human obesity. ETV5 is expressed in insulin producing pancreatic beta cells and its deletion in Etv5-/- mice results in less weight gain during high fat diet and impaired glucose tolerance due to reduced insulin exocytosis and less β-cell mass. Against this background we investigated whether ETV5 is regulated by nutritional states in pancreatic beta cells and whether ETV5 plays a role in proliferation and survival during diabetogenic stresses. Insulin producing rat insulinoma-derived INS-1E cells and generated shEtv5-INS1E cells with Dox-inducible Etv5 knockdown served as models. The obtained results demonstrate that ETV5 production is not regulated by low and high glucose or serum deprivation. ETV5 is time and dose-dependently induced by streptozotocin (STZ) and palmitate (PA) to significant levels after 24 h while alloxan has no effect. STZ and PA induce Etv5 gene expression also in primary mouse islets. Knockdown of Etv5 did not change proliferation in expanding INS-1E β-cells but significantly further enhanced STZ- and PA-mediated loss of viability despite same activation of apoptotic effector caspases-3/7 as in control cells. Instead, Etv5 knockdown increased NO production under basal conditions and enhanced necrotic cell death in response to STZ and PA.
In conclusion, this study provided initial evidence that ETV5 plays a currently not completely characterized role in the survival of pancreatic insulin producing β-cell during diabetogenic stress.

Disclosure
F. Liu: None. G. Päth: None. K. Laubner: Research Support; Self; Boehringer Ingelheim International GmbH, GI Dynamics, Inc. Speaker's Bureau; Self; AstraZeneca, Berlin-Chemie AG, Boehringer Ingelheim International GmbH, Merck Sharp & Dohme Corp., Novartis AG. J. Seufert: None.

Funding
Brigitte Bull Foundation; China Scholarship Council (201509370027 to F.L.)
2123-P: Peroxiredoxin6 Is a Novel Promoter of Pancreatic Beta-Cell Survival
Authors: PACIFICI FRANCESCA , DELLA-MORTE DAVID , CAPUANI BARBARA , PASTORE DONATELLA , ARRIGA ROBERTO , COPPOLA ANDREA , DONADEL GIULIA , BELLIA ALFONSO , LAURO DAVIDE 
Published: June 2019

Background: Diabetes is a multifactorial disease with low levels of inflammation and loss of functional pancreatic β-cell mass. Pancreatic β-cells have a reduced antioxidant capacity with an increased vulnerability to oxidative stress. Pro-inflammatory cytokines increasing reactive oxygen species production lead to cell death. Peroxiredoxin6 (Prdx6), an antioxidant enzyme, is highly expressed in beta cells compared to other antioxidants. We previously reported as Prdx6 knockout (Prdx6-/-) mice have a mild diabetes mellitus phenotype due to a reduction of pancreatic β-cell mass. Moreover, the higher levels of TNFα in Prdx6-/- mice may induce β-cell loss, contributing to the reduction in β-cell mass. However, the intracellular apoptotic pathway leading to cell death have not been investigated, yet.
Aims and Results: The objective of this study was to analyze the role of Prdx6 in the modulation of apoptosis in response to TNFα. To achieve this goal, Prdx6 was stably silenced (Prdx6KD) in murine pancreatic beta cell line (βTC6), and, the activation of intrinsic and/or extrinsic apoptotic pathway in response TNFα was analyzed in Prdx6KD and control (Scramble) cell lines. We demonstrated that TNFα administration (50 ng/ml for 24h), induced a two folds increase in apoptosis evaluated by FACS analysis, in Prdx6KD cells compared to scramble cells. Furthermore, by using ELISA assay, we measured the activity of caspase 2, 8, and 10 for the extrinsic pathway and caspase 9 for the intrinsic one. We found that after TNFα treatment, Prdx6KD cells exhibited significant higher levels (p&lt;0.05) of caspase 8 and 9 compared to control cells. Moreover, also the activation of caspase 3 and PARP1 cleavage were significantly augmented in Prdx6KD cells compared to scramble cells, both treated with TNF-α as reported above.
Conclusion: These results suggest that Prdx6 can regulate pancreatic β-cells apoptosis.
In conclusion, Prdx6 can be considered a novel potential therapeutic target for diabetes, preserving pancreatic β-cells survival.

Disclosure
F. Pacifici: None. D. Della-Morte: None. B. Capuani: None. D. Pastore: None. R. Arriga: None. A. Coppola: None. G. Donadel: None. A. Bellia: None. D. Lauro: None.
2135-P: Genetic Ablation of CB1R Protects against Streptozotocin-Induced ß-Cell Destruction in Mice
Authors: ASEER KANIKKAI RAJA , EGAN JOSEPHINE M. 
Published: June 2019

Approximately 1.25 million Americans have type 1 diabetes mellitus (T1DM), resulting from β-cell destruction. There is no known way to prevent it or to stop destruction of any remaining β-cells. Although the cannabinoid system, specifically cannabinoid 1 receptor (CB1R), is implicated in obesity and T2DM, its role, if any, in T1DM has not been studied. To address this, we induced β-cell destruction in wild type mice (WT: n=8) and mice in which CB1R was genetically nullified in β-cells only (β-CB1R-/-: n=8) in adulthood by five injections of streptozotocin (50 mg/kg intraperitoneally [IP]), a toxin selective for β-cells. We found that β-CB1R-/- mice had random blood glucose (BG) levels in the range of 137-289 mg/dL throughout the study period (28 days) as well as preserved insulin secretion with superior glucose tolerance after IP glucose (2g/kg) at day 8, 15 or 27, relative to their (random BG 500-600 mg/dL) WT littermates. CB1R knockout drove significant increases in pancreatic islet autophagic activity based on increased accumulation of autophagosome hallmarks (LC3B, Beclin-1, ATG5 and ATG7) and reduced p62 expression. Insulin and LC3B were colocalized in CB1R-deficient β-cells. Remarkably, the islets did not show the upregulation of ER unfolded protein response, which was present in β-cells of WT mice, while exerting the induction of protective chaperones. These anti-stress functions were accompanied by increased phosphorylation of AKT (ser) 473 and ERK in β-cells, which reveals a previously unrecognized route for PI3K-AKT pathway to activate the anti-apoptotic NFkB/p65 signaling that coordinately is permissive to β-cell survival. We conclude that CB1R nullification restores autophagy machinery as a novel signaling pathway to counteract ER stress-mediated cell death and confers resistance to β-cell destruction. We propose that CB1R is a potential therapeutic target during the honeymoon phase of T1DM.

Disclosure
K. Aseer: None. J.M. Egan: None.
2137-P: Cholecystokinin Protects Mouse Pancreatic Beta Cells against Cytokine Insult through the Cholecystokinin A Receptor
Authors: KIM HUNG TAE , SACOTTE STEVE , WILLIAMS RASHAUN A. , DE SOUZA ARNALDO H. , HAN JEE YOUNG , BARTOSIAK JACOB T. , YANG GRACE H. , DAVIS DAWN B. 
Published: June 2019

Cholecystokinin (CCK) is an incretin-like hormone that is also produced by pancreatic β-cells under conditions of stress and obesity. In the past, we have established a role for CCK in protection from β-cell apoptosis. However, the specific CCK receptor responsible for this protective effect was unknown. Both Cckar and Cckbr mRNA are expressed in mouse islet, although Cckbr expression is very low. Incubation of WT mouse pancreatic islets with CCK-8 (100nM) increases the transcript levels of Cckar but does not impact Cckbr expression. Conversely, 24-hour treatment with proinflammatory cytokine cocktail decreases CCKAR transcript levels but again does not modulate Cckbr expression. Therefore, we hypothesized that the CCKA receptor was the most likely mediator of CCK signaling in the β-cell. We used isolated islets from mice with germline knockout of CCK receptors to determine which receptor was required for the pro-survival effects of CCK on the β-cell. Using imaging flow cytometry of dissociated islet cells stained for annexin V and propidium iodide (PI) we find that islet cells from CCKBR knockout mice have a 19.3% (p&lt;0.005) reduction in cytokine-mediated cell death when treated with CCK-8 (100 nM), similar to the protection seen in wild type islets. This protective effect is lost in the CCKAR and double knockout mouse islets. We also confirm this finding using insulin and TUNEL immunohistochemistry to specifically identify β-cell apoptosis. In CCKBR knockout mouse islets, CCK-8 (100 nM) reduces the % TUNEL positive β-cells from 12.98% to 4.39% (p&lt;0.05) after cytokine exposure, similar to protection seen in WT mouse islets (10.47% to 4.48%). Again, CCK-8 does not protect β-cells from cytokine-induced apoptosis in CCKAR knockout or double CCKR null mouse islets. Taken together, CCKAR, but not CCKBR, is required for CCK-mediated protection of pancreatic β-cells from cytokine-induced stress.

Disclosure
H. Kim: None. S. Sacotte: None. R.A. Williams: None. A.H. de Souza: None. J. Han: None. J.T. Bartosiak: None. G.H. Yang: None. D.B. Davis: None.

Funding
National Institutes of Health
2143-P: GLP-1 Conjugation Enhances the Activity of Antisense Oligonucleotides Targeting Islet Amyloid Polypeptide (IAPP) or Kelch-Like ECH Associated Protein 1 (Keap1) in Isolated Pancreatic Islets
Authors: FU WUXIA , LEE RICHARD , PIRIE ELAINE C. , PRAKASH THAZHA , MURRAY SUE , POWERS ANDREW F. , CROOKE ROSANNE M. 
Published: June 2019

The incidence of type 2 diabetes (T2D) is increasing rapidly and is currently one of the 21st century’s major challenges to human health. While T2D has a complex etiology, beta cell dysfunction is widely regarded as a key contributor to T2D disease progression. Antisense oligonucleotides (ASOs) have been shown to provide substantial therapeutic benefit for numerous unmet medical needs. However, unconjugated ASOs have shown minimal activity in pancreatic beta cells, limiting their use as therapeutics in these cells. It has recently been shown that conjugation of glucagon like peptide 1 receptor (GLP-1R) ligands (GLP-1R-L) to ASOs led to significant, substantial increases of ASO activity in the pancreatic islet beta cell, making them a viable therapeutic option for treatment of beta cell dysfunction.1 Two factors that contribute to beta cell dysfunction are IAPP and oxidative stress. A key inhibitor of the intracellular anti-oxidative response is Keap1. We treated isolated islets with parent or GLP-1R-L conjugated ASOs targeting either Keap1 or IAPP. At the highest dose, the parent ASOs reduced their respective islet target mRNAs by up to 20%, while the GLP-1 conjugated ASOs reduced target mRNA by 85%-95%. Furthermore, the GLP-1R-L conjugated Keap1 ASO treatment increased mRNA expression of the anti-oxidative response gene NAD(P)H quinone dehydrogenase 1 (Nqo1) by 390% when compared to PBS controls. Systemic treatment of C57BL/6 mice with the GLP-1R-L conjugated IAPP ASO reduced islet IAPP mRNA by 79% ± 3.7% when compared to saline treated animals. These data suggest that GLP-1R-L conjugated ASOs show exciting potential as a therapeutic platform for the treatment of beta cell dysfunction.

Disclosure
W. Fu: None. R. Lee: Employee; Self; Ionis Pharmaceuticals, Inc. E.C. Pirie: Employee; Self; Ionis Pharmaceuticals, Inc. T. Prakash: Employee; Self; Ionis Pharmaceuticals, Inc. S. Murray: Employee; Self; Ionis Pharmaceuticals, Inc. A.F. Powers: Employee; Self; Ionis Pharmaceuticals, Inc. Stock/Shareholder; Self; Ionis Pharmaceuticals, Inc. R.M. Crooke: Employee; Self; Ionis Pharmaceuticals, Inc. Employee; Spouse/Partner; Ionis Pharmaceuticals, Inc.