bluuebunny/crossref_metadata_2025 huggingface dataset:
244-OR: Signs of Glucagon Resistance after a Two-Week Hypercaloric Diet Intervention
Authors: SUPPLI MALTE P. , BAGGER JONATAN I. , HOLST JENS J. , KNOP FILIP K.
Published: June 2023

Hyperglucagonemia is observed in individuals with obesity and contributes to the hyperglycemia of persons with diabetes. Hyperglucagonemia may arise due to reduced hepatic amino acid turnover and ensuing elevations in glucagonotropic amino acids. We recently observed reduced glucagon sensitivity and hyperglucagonemia in individuals with obesity and hypothesized it to be a consequence of steatosis. Here, we evaluated whether reduced glucagon sensitivity could be induced by a short-term hypercaloric diet intervention designed to increase hepatic fat content in healthy individuals.

We recruited 20 healthy, lean individuals (BMI: 23 ± 0.3 (mean ± SD) kg/m2) to follow a hypercaloric diet (~5,000 kcal/day) and a sedentary lifestyle for 2 weeks. Amino acid turnover in response to infusion of glucagon was assessed during a pancreatic clamp with somatostatin and basal insulin. Participants were examined before and after the lifestyle-intervention and again after 8 weeks to assess the reversibility of any metabolic changes. Hepatic steatosis was assessed by magnetic resonance spectroscopy.

The intervention led to increases in body weight (3.5 [2.8;4.2] (mean [95% confidence interval]) kg, P < 0.0001) and hepatic fat content (382 [206;705]%, P < 0.001). Insulin resistance was evident with unchanged fasting blood glucose levels and a 56% increase in fasting insulin (P < 0.001). Glucagon infusion led to a decrease in the concentration of total amino acids, but the percentage change in total amino acids was reduced (−2.5 ± 0.5 vs. −0.2 ± 0.7%, P = 0.015) and the average slope of the total amino acid curve was less steep (−2.0 ± 1.2 vs. −1.2 ± 0.3 μM/min, P = 0.016) after the intervention compared to baseline. All metabolic changes were normalized at follow-up.

Our results indicate that short-term unhealthy behavior increasing hepatic fat content causes a reversible resistance to the effect of glucagon on amino acid turnover in healthy individuals, which may explain hyperglucagonemia associated with obesity and diabetes.

Disclosure

M.P.Suppli: None. J.I.Bagger: Speaker's Bureau; Novo Nordisk A/S. J.J.Holst: Advisory Panel; Novo Nordisk A/S, Eli Lilly and Company, Board Member; Bainan Biotech, Antag Therapeutics, Consultant; Alphasights, Eli Lilly and Company, ShouTi Pharma Inc., Zealand Pharma A/S, Other Relationship; Novo Nordisk, Novo Nordisk Pharma, Mayo Clinic, Boehringer-Ingelheim, Scohia Pharma Inc., Research Support; ARLA, European Union, Novo Nordisk Foundation. F.K.Knop: Advisory Panel; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Novo Nordisk, Sanofi, Consultant; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Novo Nordisk, Sanofi, Research Support; Novo Nordisk, Zealand Pharma A/S, Speaker's Bureau; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Novo Nordisk, Sanofi, Lundbeck.
246-LB: Stimulation of Hepatic Lipid Secretion by Glucocorticoid Action in the Nucleus of the Solitary Tract
Authors: VASILEV BOYAN , GREWAL MANTASH S. , LUM BRYAN , YUE JESSICA T.
Published: June 2023

Dysregulated lipid metabolism is a characteristic of diabetes, insulin resistance, and obesity. Increased glucocorticoid (GC) levels and/or action is associated with elevated levels of circulating triglyceride (TG)-rich very low-density lipoproteins (VLDL-TGs). Lipid metabolism is heavily affected by GCs via its direct actions on peripheral organs, e.g. liver, adipose tissue. However, how GCs modulate hepatic VLDL-TG secretion via their actions in the brain is less known. The nucleus of the solitary tract (NTS) in the hindbrain is responsive to various hormones and is an important region involved in whole-body metabolism. Here, we aimed to assess how GC action in the NTS affects hepatic TG secretion and hypothesized that GC receptor (GR) agonism in the NTS stimulates hepatic TG secretion.

Sprague-Dawley rats underwent stereotaxic NTS bilateral cannulation and vascular catheterizations to enable simultaneous direct NTS infusions, intravenous injections, and blood sampling. Hepatic TG secretion rates were measured in 10h-fasted rats after intravenous poloxamer injection with concurrent NTS infusions. NTS GC infusion stimulated TG secretion compared to NTS vehicle controls, but this effect was negated with pharmacological and genetic GR antagonism, suggesting that NTS GCs are mediated via its receptor. The NTS GC-induced increase in VLDL-TG was not associated with changes in liver lipogenic gene expression and protein levels or plasma ApoB48/100. However, plasma FFAs were increased in NTS GC infused rats, and this was associated with increased pHSL:HSL protein levels in white adipose tissue.

We herein provide evidence that acute GC action in the NTS stimulates hepatic VLDL-TG secretion and affects adipose tissue lipid metabolism. Increased circulating FFA availability may contribute to the hyperlipidemic effects of NTS GCs. Future studies to assess the role of NTS GC action in diabetic and obesity-related metabolic disease dyslipidemic models are warranted.

Disclosure

B. Vasilev: None. M. S. Grewal: None. B. Lum: None. J. T. Yue: None.

Funding

Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-06657); Canada Foundation for Innovation (37267); Canada Research Chairs Tier 2 in Brain Regulation of Metabolism
248-LB: Aging of Tanycytes Abolishes the Dynamic Regulation of Blood–Brain Barrier Permeability in the Hypothalamus
Authors: PHAM VIANA , XU ALLISON , BACHOR TOMAS P.
Published: June 2023

Aberrant metabolic regulation and blood-brain barrier (BBB) dysfunction are two well-known phenomena of aging. The hypothalamus is the main brain region that controls food intake and body weight. It contains a specialized BBB that allows for the free flow of blood through fenestrated capillaries in the median eminence (ME), which places a portion of neurons critical to feeding unprotected by the BBB. Aging's pernicious effect on the traditional BBB throughout the brain is well studied, however how the specialized BBB in the hypothalamus is altered with age is unknown. To study the effects of aging on hypothalamic injury and repair, we injected monosodium glutamate (MSG) into young and old mice to ablate neurons located outside the BBB. We found that in the hypothalamus of young animals, MSG led to tightening of the BBB imparted by a reduction in the number of fenestrated capillaries. In contrast, aged mice were unable to dynamically alter the BBB permeability or modulate fenestrated blood vessels in response to MSG injury. We show that hypothalamic tanycytes, radial glial-like cells lining the wall of the third ventricle, show a higher degree of physical contact with fenestrated blood vessels than with non-fenestrated blood vessels. Tanycytes in young mice following MSG exposure, modified their expression of pleiotrophin, a growth and angiogenic factor, and robustly proliferated. In comparison, tanycytes in aged mice lacked in these responses to injury, which indicate these cells undergo functional decline with aging. Moreover, aged mice following MSG lost a greater number of neurons outside the BBB and experienced severe weight loss weeks after treatment, whereas young mice showed no change in their body weight. These findings uncover that aging leads to loss of hypothalamic BBB plasticity and defective tanycytes, which increase hypothalamic vulnerability to injury and morbidity development in aged animals.

Disclosure

V. Pham: None. A. Xu: Advisory Panel; Pennington Biomedical Research Center, Research Support; Eli Lilly and Company. T. P. Bachor: None.

Funding

National Institutes on Aging (1R01AG063506-01)
248-OR: Diabetes Remission after Gastric Bypass Is Characterized by Upregulation of LXR and Antioxidant-Related Pathways in Jejunum
Authors: STEFATER-RICHARDS MARGARET A. , GOURASH WILLIAM F. , COURCOULAS ANITA
Published: June 2023

We have previously shown that intestinal adaptation precedes and relates to metabolic improvement in humans after Roux-en-Y gastric bypass surgery (RYGB). Based on this, we hypothesized that persistence of this adaptation at the one-year postoperative timepoint occurs selectively in patients with persistent postoperative metabolic improvement, versus those without Type 2 diabetes (T2D) remission. RNA-seq was used to determine gene expression in jejunum collected before and 12 months (± 1 month) after RYGB. T2D was defined as HbA1c >6.5% or requiring T2D medications prescribed for prior diagnosis of diabetes. Remission was defined as HbA1c <6.5% off any T2D medications. Nine of 17 patients with T2D failed to achieve remission by 12 months. After sample quality control, jejunal biopsies from 7 patients with T2D without remission (NR), 8 patients with diabetes remission (R) and 13 patients without diabetes (ND) were analyzed via RNA-seq. Significance was defined by an FDR cutoff of 0.05. Unsupervised principal components analysis (PCA) showed no significant differences across PC1 or PC2 among the 3 groups. However, among top pathways expressed in samples from patients with T2D remission (R), LXR/RXR pathway activation was upregulated (pathway z-score= 0.333, p= 4.85E-06) but downregulated in patients without diabetes (ND, z-score= -1.508, p=.01) and in patients without remission (NR, z-score=-1.0, p=2.07E-07). Antioxidant action of vitamin C exhibited a similar pattern of activation uniquely in R and not in NR or ND (R: p=.02, z-score=0.816; NR: p=0.33, z-score=0.816; ND: p=0.03, z-score=-1.0). In summary, although jejunal remodeling may occur in patients without T2D remission, lipogenic (LXR-driven) and antioxidative pathways are uniquely upregulated in biopsies from those with remission. We hypothesize that these processes may underlie mechanisms for T2D improvement after RYGB.

Disclosure

M.A.Stefater-richards: None. W.F.Gourash: None. A.Courcoulas: None.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (R56DK125816, R01DK108642, K08DK125878)
251-LB: Association of Plasma Sphingolipids with Insulin Secretion and Insulin Sensitivity in Healthy Black and White Adults
Authors: ASUZU PEACE , MANDAL NAWAJES , WAN JIM Y. , STENTZ FRANKIE B. , DAGOGO-JACK SAMUEL
Published: June 2023

Objective: Sphingolipid (SPL) are associated with glucose dysregulation. Here, we examined plasma SPL profiles in relation to insulin action and secretion in Black and White adults.

Methods: Baseline plasma levels of 58 SPLs (ceramides {CER}, monohexosyl-ceramides {MHC}, sphingomyelins {SM}, sphingosine {SO} and dihydro-SO-1-phosphate {DHS1P}) were assayed using LC-MS/MS. Insulin sensitivity was assessed by euglycemic clamp (ISI) and HOMA-IR; insulin secretion by IVGTT (AIR), HOMA-B and disposition index (DI, i.e, AIR × ISI).

Results: We enrolled healthy offspring of parents with T2DM (75 Black, 65 White; mean age 47.7 ± 9.01 y, BMI 30.4 ± 6.12 kg/m2, FPG 92.9 ± 6.86 mg/dl and 2-hrPG 130 ± 28.8 mg/dl. Total SPLs showed no association with ISI or HOMA-IR but was significantly associated with insulin secretion (AIR, r=0.19, P=0.031; DI r=0.21, P=0.038). There were differential associations between individual SPLs and insulin secretion/sensitivity, but 5 of 58 SPLs (SM C16:0, SM C28:0, SM C28:1, SM C30:1 and SM C34:0) showed concurrent associations with both measures (r=0.16-0.37, P=0.04-<0.0001). Four SPLs were significantly associated with insulin secretion corrected for insulin sensitivity: MHC C14:0 (r=0.24, P=0.016); MHC C26:0 (r=0.23, P=0.025); SM C22:0 (r=0.26, P=0.009); DHS1P (r=0.27, P=0.006). Very long chain SMs correlated with BMI, waist circumference, body fat and lean mass (r=0.19-0.25, P=0.03-0.0004). Plasma CERs and SMs showed significant associations with 2hrPG (r=0.17-0.25, P=0.04-0.003) but not FPG.

Conclusion: Circulating SPLs associate differentially with indices of insulin sensitivity, secretion, and adiposity. Of the 58 individual SPLs assayed, MHC C14:0 and C26:0, SM C22:0 and DHS1P showed the strongest association with insulin secretion corrected for insulin sensitivity.

Disclosure

P. Asuzu: None. N. Mandal: None. J. Y. Wan: None. F. B. Stentz: None. S. Dagogo-jack: None.
252-LB: Dectin-2 Deficiency Promotes Proinflammatory Cytokine Release from Macrophages and Impairs Glucose-Stimulated Insulin Secretion
Authors: FUJITA MASAMICHI , MIYAZAWA TAKASHI , UCHIDA KEIICHIRO , UCHIDA NAOHIRO , HATAYAMA TOMOMI , NAKAMURA SHINTARO , TAKEICHI YUKINA , MIYACHI YASUTAKA , OGAWA YOSHIHIRO
Published: June 2023

Pancreatic islet inflammation plays an important role in the pathogenesis of diabetes mellitus. Islet macrophages, which have emerged as a key player in islet inflammation, express Dectin-2 (D2), one of the C-type lectin receptors recognizing α-mannans, while endocrine cells such as α-cells and β-cells lack the expression of D2. To elucidate the role of D2 in glucose metabolism and β-cell function, we analyzed the phenotype of D2 knockout (D2KO) mice. D2KO mice exhibited significantly impaired glucose tolerance compared to wild-type (WT) mice, although there were no significant differences in insulin sensitivity. Glucose-stimulated insulin secretion (GSIS) was significantly reduced in isolated islets from D2KO mice compared to WT mice. Comprehensive analysis of RNA-seq in isolated islets from D2KO and WT mice revealed that D2 deficiency induced immune cell infiltration and inflammation in pancreatic islets. Furthermore, RNA-seq analysis in peritoneal macrophages from D2KO and WT mice showed that macrophages from D2KO mice acquired an inflammatory phenotype and exhibited enhanced NF-κB signaling compared to those from WT mice. Next, to determine whether humoral factors secreted from macrophages affect GSIS, we examined GSIS using the culture supernatants of macrophages from D2KO and WT mice. The culture supernatants of macrophages from D2KO mice suppressed GSIS compared to WT mice. Furthermore, the multiplex cytokine assay revealed that IL-1α and IL-6 were significantly increased in the culture supernatants from macrophages of D2KO mice. Finally, we found that the stimulation of IL-1α and IL-6 reduced GSIS in β-cells. These findings suggest that macrophages from D2KO mice suppress GSIS by increasing IL-1α and IL-6 production. This study provides new insights into the mechanism by which D2 is involved in the pathogenesis of pancreatic islet inflammation.

Disclosure

M. Fujita: None. T. Miyazawa: None. K. Uchida: None. N. Uchida: None. T. Hatayama: None. S. Nakamura: None. Y. Takeichi: None. Y. Miyachi: None. Y. Ogawa: None.
254-OR: Effects of Roux-en-Y Gastric Bypass on Postprandial Glucose Turnover Assessed through the Single-Tracer Oral Minimal Model
Authors: BRUNASSO ALESSANDRO , DALLA MAN CHIARA , HERZIG DAVID , BALLY LIA , SCHIAVON MICHELE
Published: June 2023

Rearrangements of gastrointestinal anatomy after Roux-en-Y gastric bypass (RYGB) surgery alter postprandial glucose metabolism. However, the impact on different components of glucose homeostasis remain incompletely understood.

Here, we aim to evaluate the effects of RYGB on postprandial glucose turnover, production (P) and disposal (D) glucose effectiveness (GEP, GED) and production and disposal sensitivity to insulin (SIP, SID).

Ten RYGB (2M, age=39.2±3.1 y, BMI=28±1 kg/m2- mean±SE) subjects and 10 healthy controls (5M, age=36.4±3.2 y, BMI=26±1 kg/m2) ingested 60g of glucose (G) labelled with [6,6-2H2]-glucose. Plasma samples were frequently drawn to measure G, insulin (I) and tracer concentrations in the following 180 min. The Single-Tracer Oral Minimal Model was used to estimate meal glucose rate of appearance (Ra), endogenous glucose production (EGP), glucose disposal (Rd), GEP, GED, SIP and SID.

Incremental area under the G curve (iAUC(G)) was not different in the two groups, while iAUC(I) was significantly higher in RYGB (8346±803 vs. 5451±602 μU/mL*min, p=0.011); iAUC(Ra) normalized to the G dose was significantly higher in the first 60 min after the meal (0.72±0.04 vs. 0.28±0.03, p<0.001) while, throughout the experiment, EGP suppression (EGPs) normalized to basal (EGPb) was significantly lower (0.31±0.06 vs. 0.55±0.05, p=0.006) and iAUC(Rd) normalized to basal (Rdb) was higher (3.46±0.10 vs. 2.82±0.08, p<0.001) in RYGB than controls. Model predicted SIP was significantly lower in RYGB than controls (2.0±0.5 vs. 5.1±1.2 10-4 dL/kg/min per μU/mL, p=0.033) while no differences were observed in GEP, GED and SID.

We concluded that the abnormally high I concentration in RYGB was responsible for the higher iAUC(Rd) in the operated individuals, while the reduced SIP likely works as a protective mechanism against postprandial hypoglycaemia by reducing EGPs despite the high circulating I.

Disclosure

A.Brunasso: None. C.Dalla man: Research Support; Sanofi-Aventis Deutschland GmbH, Becton, Dickinson and Company. D.Herzig: None. L.Bally: Research Support; Dexcom, Inc., Ypsomed AG. M.Schiavon: None.

Funding

Italian Minister for Education (Law 232/2016); Swiss National Science Foundation (PCEGP3_186978)
256-LB: An OGT–TFF2 Axis in the Pathogenesis of Nonalcoholic Steatohepatitis
Authors: ZHANG LEI , WANG QI , DESROULEAUX REINA , YANG XIAOYONG
Published: June 2023

Nonalcoholic fatty liver disease (NAFLD) is a global metabolic disease that comprises a broad spectrum of liver dysfunction ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) with fibrosis. It is largely unknown how the communication between hepatocytes and non-parenchymal cells dictates the disease progression from NAFL to NASH and fibrosis. Dysregulation of protein O-GlcNAcylation in hepatocytes has been implicated in a wide range of liver diseases. In this study, we demonstrate the role of O‑GlcNAc transferase (OGT) in mediating intercellular crosstalk between hepatocytes and immune cells in NASH-fibrosis development. RNA-sequencing analysis of wildtype and hepatocyte-specific OGT knockout mice was performed and trefoil factor 2 (TFF2) was identified as one of the highly induced genes encoding secreted proteins in OGT-deficient hepatocytes. Immunohistochemistry and western blot were performed to reveal decreased OGT and increased TFF2 expression in the liver of NAFLD patients. Mechanically, OGT was found to repress TFF2 transcription in hepatocytes by modifying forkhead box protein A2 (FOXA2). Recombinant TFF2 along with NASH stimuli was applied to a 3D spheroid culture system comprised of hepatocytes and non-parenchymal cells and TFF2 was shown to promote NASH stimuli-induced triglyceride secretion and elevate the expression of pro-inflammatory and fibrogenic genes. ELISA and RT-qPCR were performed to further show that TFF2 treatment induced the expression of Th1-type cytokines (IFN-γ and TNF-α) and Th17-type cytokine (IL17) in CD4 T cells, promoting Th1 and Th17 differentiation. Collectively, these results identify an OGT-TFF2 axis that mediates the crosstalk between hepatocytes and CD4 T cells during NASH pathogenesis, revealing a potential therapeutic target for the treatment of chronic liver disease.

Disclosure

L. Zhang: None. Q. Wang: None. R. Desrouleaux: None. X. Yang: None.

Funding

American Diabetes Association (1-19-IBS-119 to X.Y.); National Institutes of Health (R01DK089098, P30DK34989)
259-LB: Loss of AMPK Induces Elongation of Preexisting Hepatic Fatty Acids, without Affecting DNL, during Fasting–Refeeding
Authors: FLETCHER JUSTIN A. , DEJA STANISLAW , BURGESS SHAWN C. , FU XIAORONG , INIGO MELISSA MAE R. , BROWNING JEFFREY , KUCEJOVA BLANKA
Published: June 2023

AMP-activated protein kinase (AMPK) is a cellular energy sensor that conserves energy by inhibiting anabolic processes, such as de novo lipogenesis (DNL). Paradoxically, fructose metabolism activates AMPK by reducing the energy state (elevated AMP/ATP ratio), yet also promotes DNL. However, the comprehensive effects of fructose metabolism and AMPK activity on hepatic DNL has not been thoroughly investigated. Here, we examine how loss of AMPK function impacts DNL in response to fasting followed by refeeding a fructose or chow diet. After a 16 hour fast, 12 liver-specific AMPKα1α2 knock-out mice (AMPK-KO) and 12 AMPK α1α2 lox/lox mice (control) were fed either high fructose or chow diets (6 per a group) and administered D2O drinking water for 7 hours prior to blood collection and tissue harvest. After Folch extraction, fatty acids in liver triglyceride (TG-FA) were analyzed by proton and deuterium NMR spectroscopy to determine 2H incorporation and estimate DNL, elongation and re-esterification fluxes. Body water enrichment (~3.5%) was similar between groups. After correcting for food intake and body weight, preliminary data indicates that the AMP/ATP ratio was greater in AMPK-KO liver compared to controls, but neither chow nor fructose diets affected the energy state. Overall, neither hepatic steatosis nor DNL differed between groups. Furthermore, loss of AMPK function did not affect the amount of various FA species (i.e. saturated, unsaturated, DHA and ω3, linoleic, and non-essential). Instead, AMPK-KO liver had increased TG-FAs that were elongated compared to controls. This effect also tended to be greater in fructose fed mice (AMPK-KO and controls) compared to chow fed mice, even though fructose fed mice consumed fewer calories.

In conclusion, loss of AMPK function resulted in increased elongation of hepatic TG-FAs, but not DNL, or the amount of FAs in the liver in response to fasting and refeeding with either fructose or chow diets.

Disclosure

J. A. Fletcher: None. S. Deja: None. S. C. Burgess: None. X. Fu: None. M. R. Inigo: None. J. Browning: None. B. Kucejova: None.

Funding

National Institutes of Health (K01AA030327)
264-OR: Incorporating Shared Decision-Making to Facilitate Equitable Insulin Pump Use among Pediatric Patients with Type 1 Diabetes
Authors: TURNER JOEL , NELSON GRACE , ADAMS BLAKE V.
Published: June 2023

Background: Insulin pumps, CGMs, and other technologies have shown to improve outcomes for patients with T1D. However, various obstacles prevent some patient groups from utilizing these technologies. This was evident by the discrepancy in non-Hispanic black patients on insulin pumps (9%) vs non-Hispanic white patients (23%) in our clinic. Our goal is to identify and overcome these obstacles in order to safely increase the total number of patients that utilize insulin pumps while also creating equity in pump use between groups.

Methods: We identified a group of non-Hispanic black patients who were not candidates for starting insulin pumps based on our clinic’s “typical” practice, but showed interest in pumps. By focusing on this group, we continued to adjust pump prescriptions and processes. After demonstrating efficacy and safety of pump use in this group, we began expanding to all patients as appropriate. Beyond continued education for nurse educators and changing pump start criteria, we also started using shared decision-making to be sure all patients were learning about technology on a regular basis.

Results: As of December 2022, we have helped >35% of our interested patients navigate the process of obtaining an insulin pump. Equity was also improved, with 20% of our non-Hispanic black patients now using pumps vs 25% of non-Hispanic white patients. Some of these patients would not typically be candidates for pumps but were able to safely begin using this technology because of the interventions we initiated.

Conclusions: Utilizing shared decision-making can help to identify patients who might not initially be considered for insulin pumps and other technology. We have demonstrated that many interested patients can safely initiate pumps which will help improve management of their diabetes. We are continually adjusting our processes to improve equity in pump use and increase the overall number of patients utilizing pumps in our clinic.

Disclosure

J.Turner: None. G.Nelson: None. B.V.Adams: None.
266-LB: SGLT2 Inhibition Improved Endurance Performance of db/db Mice Accompanied by an Increased Endogenous AMPK Activator in Skeletal Muscles
Authors: NAKAMURA SHINTARO , MIYACHI YASUTAKA , YOKOMIZO HISASHI , OTSUKA HIROKO , SAKAMOTO RYUICHI , TAKAHASHI MASATOMO , IZUMI YOSHIHIRO , MIYAZAWA TAKASHI , BAMBA TAKESHI , OGAWA YOSHIHIRO
Published: June 2023

Background: Diabetes is associated with loss of skeletal muscle mass and function. Specifically, diabetes may affect the function of oxidative muscle fibers with lower fatigability and higher oxidative capacity in skeletal muscle. The effects of SGLT2 inhibition on skeletal muscle metabolism and function in patients with type 2 diabetes remain unclear.

Methods: Male db/db (diabetic) and db/+ (non-diabetic) mice at 8 weeks of age were fed a normal chow diet or a diet containing canagliflozin (CANA) for 4 weeks. Grip strength and running distance were assessed during CANA treatment. At the end of CANA treatment, we measured muscle weight and analyzed metabolites in skeletal muscle.

Results: The weights of the oxidative soleus and glycolytic extensor digitorum longus muscles were reduced by approximately 50% in the diabetic group compared to the non-diabetic group, but neither was altered by CANA treatment. Grip strength did not differ between the CANA-treated diabetic, diabetic, and non-diabetic groups. However, the running distance decreased in the diabetic group compared to the non-diabetic group (89 ± 60 m vs. 1980 ± 276 m). This was partially restored by CANA treatment (89 ± 60 m vs. 415 ± 140 m). Metabolomic analysis revealed that long-chain acyl-CoA increased and medium-chain and short-chain acyl-CoA decreased in diabetic soleus muscle. In contrast, CANA treatment increased medium-chain and short-chain acyl-CoA. Furthermore, CANA increased 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5’-monophosphate (AICARP), an endogenous AMPK activator, by approximately 3-fold in diabetic soleus muscle with increased levels of AMPK phosphorylation (Thr172).

Conclusion: These results suggest that SGLT2 inhibitors may activate the AICARP/AMPK pathway and improve impaired oxidative muscle function in patients with type 2 diabetes.

Disclosure

S. Nakamura: None. Y. Miyachi: None. H. Yokomizo: None. H. Otsuka: None. R. Sakamoto: None. M. Takahashi: None. Y. Izumi: None. T. Miyazawa: None. T. Bamba: None. Y. Ogawa: None.
267-OR: ADA Presidents' Select Abstract: Combining an SGLT Inhibitor with a Glucagon Receptor Agonist (GRA) in Subjects with Type 1 Diabetes—A Randomized Clinical Trial
Authors: BOEDER SCHAFER C. , ROUX MELISSA J. LE , GIOVANNETTI ERIN R. , ARMSTRONG ADRIENNE , CARTER LESLIE , THOMAS ROBERT L. , GREGORY JUSTIN M. , PETTUS JEREMY
Published: June 2023

SGLTi treatment improves glycemic control and reduces insulin dosing but increases the risk of diabetic ketoacidosis (DKA). Combination therapy (SGLTi + GRA) could augment glycemic and insulin dosing benefits while mitigating DKA risk.

Subjects with T1D (n=12) received 4-week insulin-adjunctive treatments with (a) SGLTi (dapagliflozin 10 mg) + placebo and (b) SGLTi + GRA (volagidemab 70 mg) in a cross-over, double-blind design. At baseline and end of each treatment, CGM and insulin dosing were recorded, and subjects completed an 8-hr insulin withdrawal test (IWT).

Average glucose improved with both SGLTi (138 mg/dL; P < 0.01) and SGLTi + GRA (131 mg/dL; P < 0.01) compared to Baseline (150 mg/dL). Total daily insulin dose was reduced with SGLTi (0.52 U/kg/day) compared to Baseline (0.56 U/kg/day; P = 0.06) and was reduced further with SGLTi + GRA treatment (0.41 U/kg/day; P < 0.01 vs SGLTi). Peak beta-hydroxybutyrate (BHB) concentrations during IWT were lower with SGLTi + GRA treatment (2.03 mmol/L) than with SGLTi alone (2.44 mmol/L; P = 0.04); there was no significant difference in peak BHB with either treatment vs Baseline (2.14 mmol/L).

Treatment with SGLTi + GRA improved glycemic control, reduced insulin use, and mitigated ketosis during hypoinsulinemia. These data suggest that combination adjunctive therapy may be effective to address multiple metabolic abnormalities in T1D.

Disclosure

S.C.Boeder: Consultant; Cecelia Health, Research Support; Dexcom, Inc., REMD Biotherapeutics, Eli Lilly and Company. M.J.Le roux: None. E.R.Giovannetti: None. A.Armstrong: None. L.Carter: None. R.L.Thomas: None. J.M.Gregory: Advisory Panel; Eli Lilly and Company, vTv Therapeutics, Other Relationship; Medtronic. J.Pettus: Advisory Panel; Sanofi, Novo Nordisk, Lilly, MannKind Corporation, Consultant; Carmot Therapeutics, Inc., Diasome.

Funding

National Institutes of Health (P30DK063491, UL1TR001442); Diabetes Research Connection
27-LB: A Prediction Model for Diabetic Retinopathy Based on Plasma Adipokines among Patients with Type 2 Diabetes
Authors: AN YAXIN , KE JING , ZHAO DONG
Published: June 2023

Background: Accumulating evidence has suggested a link between adipokines and diabetic retinopathy (DR). This study aimed to investigate the risk factors for sight-threatening DR (STDR) and establish a prognostic model for predicting STDR among patients with type 2 diabetes mellitus (T2DM).

Methods: In the case-control set, principal component analysis (PCA) was performed to select optimal predictive cytokines for STDR. Support vector machine (SVM) was used to examine the possible combination of baseline plasma adipokines to discriminate the mild participants who will later develop STDR. An individual prospective cohort with a follow-up period of 3 years was used for the external validation.

Results: In both training and testing sets, comprised of 306 patients with DR, median levels of plasma adiponectin, leptin, and fatty acid-binding protein 4 (FABP4) were significantly higher in the STDR group than those in mild DR. Except for adipsin, the other three adipokines, FABP4, adiponectin, and leptin, were selected by PCA and integrated into SVM. The accuracy of the multivariate SVM classification model was acceptable in both the training set (AUC = 0.81, sensitivity = 71%, specificity = 91%) and the testing set (AUC = 0.77, sensitivity = 61%, specificity = 92%). 110 T2DM mild NPDR patients were enrolled for external validation. High- and low-risk groups were grouped by the median value of adiponectin, FABP4, and leptin. More STDR occurred in the high-risk group than in the low-risk group among the three predictors. SVM demonstrated acceptable performance, with the AUC, sensitivity, and specificity of 0.77, 64%, and 91%, respectively.

Conclusions: Adiponectin, leptin, and FABP4 were demonstrated to be associated with the severity of DR and maybe good predictors for STDR, suggesting that adipokines may play an important role in the pathophysiology of DR development.

Disclosure

Y. An: None. J. Ke: None. D. Zhao: None.
271-OR: Capillary Blood Ketone Level and Future Ketoacidosis Risk in Type 1 Diabetes Using Sodium–Glucose Linked Transporter Inhibitors
Authors: BAPAT PRIYA , DHALIWAL SHARON , SONG CIMON , BUDHRAM DALTON R. , BAKHSH ABDULMOHSEN M.K. , SCARR DANIEL , WEISMAN ALANNA , FRALICK MICHAEL , IVERS NOAH , CHERNEY DAVID , MUMFORD DOUG , TOMLINSON GEORGE , ERIK LOVBLOM LEIF , PERKINS BRUCE A.
Published: June 2023

Adjunctive-to-insulin Sodium-Glucose Linked Transporter inhibitors (SGLTi) improve metabolic control in T1D but cause diabetic ketoacidosis (DKA). We aimed to determine if time-varying ketone levels could predict future DKA. We explored data on 1194 participants randomly assigned to empagliflozin in a T1D trial program (NCT02414958 and NCT02580591). Protocol called for fasted capillary blood ketone levels 2-3 times/week, categorized into maximum, mean, and last values for sequential 1-month ‘index test’ periods during 6-12-month follow-up. Trial-adjudicated DKA/Ketosis was determined in subsequent 1-month ‘outcome’ periods. Area under the Receiver Operator Characteristic curves (AROC) were calculated. Gradient-boosted trees determined if prediction could be improved. Participants had mean age 44.3 years, HbA1c 8.14±0.60 percent, and contributed 3.70±2.97 ketone measurements/week. 617 outcomes (568 ketosis, 49 DKA events) occurred over follow-up. On-treatment ketone levels were higher for index test periods preceding an outcome period with an event compared to those without (for example, maximum ketone was 1.32±1.03 vs 0.80±0.71 mmol/l, respectively, p<0.001). AROC for maximum, mean and last ketone levels were 0.764, 0.755, and 0.682 respectively. Maximum ketone level ≥ 0.9 mmol/l had sensitivity 59%, specificity 84%, likelihood ratio positive 3.65, likelihood ratio negative 0.49, and diagnostic odds ratio 7.39. Inclusion of the other ketone measures (mean or last) or clinical variables did not improve the predictive validity of maximum ketone levels on their own. Routine surveillance of morning capillary ketone levels in T1D patient using SGLTi can predict 1-month risk of DKA/Ketosis and could allow for interventions to mitigate risk.

Disclosure

P.Bapat: None. D.Cherney: Other Relationship; Boehringer Ingelheim-Lilly, Merck, AstraZeneca, Sanofi, Mitsubishi-Tanabe, Abbvie, Janssen, Bayer, Prometic, BMS, Maze, Gilead, CSL-Behring, Otsuka, Novartis, Youngene, Lexicon and Novo-Nordisk, Research Support; Boehringer Ingelheim-Lilly, Merck, Janssen, Sanofi, AstraZeneca, CSL-Behring and Novo-Nordisk. D.Mumford: None. G.Tomlinson: None. L.Lovblom: None. B.A.Perkins: Advisory Panel; Dexcom, Inc., Insulet Corporation, Novo Nordisk, Sanofi, Vertex Pharmaceuticals Incorporated, Other Relationship; Abbott, Medtronic, Sanofi, Research Support; Novo Nordisk, Bank of Montreal (BMO). S.Dhaliwal: None. C.Song: None. D.R.Budhram: None. A.M.K.Bakhsh: None. D.Scarr: None. A.Weisman: None. M.Fralick: None. N.Ivers: Consultant; Novo Nordisk Canada Inc., IQVIA Inc.

Funding

Diabetes Canada (OG-3-21-5572-BP)
272-LB: Obesity-Induced Mitochondrial Fragmentation in White Adipocytes Is Controlled by the Small GTPase RalA
Authors: XIA WENMIN , VEERAGANDHAM PREETHI , CAO YU , XU YAYUN , RHYNE TORREY E. , JONES YING , YU RUTH T. , RYDÉN MIKAEL , WABITSCH MARTIN , SALTIEL ALAN
Published: June 2023

Mitochondrial dysfunction has been implicated in the development of obesity, insulin resistance and fatty liver disease. Here we report that mitochondria undergo fragmentation and reduced oxidative capacity specifically in inguinal white adipose tissue after high fat diet, and that this process is regulated by the small GTPase RalA. RalA mRNA, protein and GTP binding activity are increased in inguinal white adipose tissue after HFD feeding. Targeted deletion of Rala in white adipocytes prevents the obesity-dependent fragmentation of mitochondria, and produces mice who are resistant to HFD-induced weight gain via increased energy expenditure, as well as improvements in glucose homeostasis and liver metabolism. In vitro studies revealed that RalA suppresses mitochondrial oxidative function in adipocytes by increasing fission through reversing the inhibitory phosphorylation of the mitochondrial fission protein Drp1. This reduced phosphorylation results from the recruitment of the regulatory subunit of protein phosphatase PP2A, which acts as a bona fide effector of RalA, leading to the specific dephosphorylation of the inhibitory Ser637 residue on Drp1, rendering the protein active. Thus, RalA plays a key role in repressing energy expenditure in obese adipose tissue through maintaining mitochondrial fission, contributing to weight gain and related metabolic dysfunction, including glucose intolerance and fatty liver, and may explain in part how energy expenditure is repressed in prolonged obesity.

Disclosure

W. Xia: None. A. Saltiel: Board Member; Elgia Therapeutics. P. Veeragandham: None. Y. Cao: None. Y. Xu: None. T. E. Rhyne: None. Y. Jones: None. R. T. Yu: None. M. Rydén: Advisory Panel; Boehringer-Ingelheim, Eli Lilly and Company, Consultant; Sigrid Therapeutics, Other Relationship; Novo Nordisk Foundation, Speaker's Bureau; Sanofi, Novo Nordisk. M. Wabitsch: None.
272-OR: The Efficacy of Sotagliflozin on Heart-Failure Related Outcomes Is Independent of Baseline A1C
Authors: AGGARWAL RAHUL , BHATT DEEPAK L. , SZAREK MICHAEL , DAVIES MICHAEL J. , BANKS PHILLIP L. , PITT BERTRAM , STEG PHILIPPE G.
Published: June 2023

Background: SGLT inhibitors reduce the risk of cardiovascular (CV) events in patients with heart failure (HF) and chronic kidney disease regardless of diabetes status. Although the SOLOIST-WHF and SCORED trials enrolled high CV risk patients with T2DM, participants had a wide range of baseline A1C values. Thus, the present analysis evaluated the effects of sotagliflozin relative to placebo on cardiovascular (CV) death and total HF-related outcomes by baseline A1C range.

Methods: SOLOIST-WHF had no baseline A1C entry criteria (baseline median = 7.1%, range = 4.5, 15.0%), while SCORED randomized patients with T2DM and an A1C ≥7% at screening (8.3%, 6.5, 17.3%). Natural cubic splines from Poisson regression models estimated the relationships between continuous A1C and the primary endpoint for both studies: total number of CV deaths, hospitalizations for HF, and urgent visits for HF.

Results: Within each trial, patients with higher baseline A1C experienced higher event rates (spline effect p=0.0349 and 0.0014 for SOLOIST-WHF and SCORED, respectively). Sotagliflozin reduced the primary outcome in each trial (p for treatment <0.0001 for both SOLOIST-WHF and SCORED) and this did not differ across A1C (p for interaction >0.10; Figure).

Conclusion: Sotagliflozin was effective in reducing the risk of CV death and HF-related events independent of baseline A1C.

Disclosure

R.Aggarwal: None. D.L.Bhatt: Board Member; Bristol-Myers Squibb Company, Research Support; Lexicon Pharmaceuticals, Inc., Sanofi, Lilly, Boehringer Ingelheim Inc., AstraZeneca, Novo Nordisk. M.Szarek: Consultant; Amarin Corporation, ESPERION Therapeutics, Inc., Other Relationship; Janssen Pharmaceuticals, Inc., Research Support; Lexicon Pharmaceuticals, Inc., Regeneron, Sanofi, Bayer Inc. M.J.Davies: Employee; Lexicon Pharmaceuticals, Inc. P.L.Banks: Employee; Lexicon Pharmaceuticals, Inc. B.Pitt: Consultant; Bayer Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Vifor Pharma Management Ltd., Lexicon Pharmaceuticals, Inc., PhaseBio Pharmaceuticals, Inc., KBP Bioscience, Merck & Co., Inc. P.G.Steg: Advisory Panel; Merck & Co., Inc., Consultant; Sanofi, Amarin Corporation, Amgen Inc., BMS, PhaseBio Pharmaceuticals, Inc., Idorsia Pharmaceuticals Ltd., Novartis AG, Janssen Pharmaceuticals, Inc., AstraZeneca, Research Support; Lexicon Pharmaceuticals, Inc.
275-LB: Mitochondrial Fission Factor Can Be a Key Factor in the Regulation of Hepatic Lipid Metabolism
Authors: TAKEICHI YUKINA , MIYAZAWA TAKASHI , OGAWA YOSHIHIRO
Published: June 2023

Nonalcoholic fatty liver disease (NAFLD) develops into nonalcoholic steatohepatitis (NASH) when fat accumulates in the liver, leading to hepatocyte apoptosis, inflammation and fibrosis. No effective treatments for NASH have been established. Mitochondria are important organelles that play a central role in de novo lipogenesis and fatty acid β-oxidation. We have shown that mitochondrial fission is enhanced by short-term high-fat diet (HFD) loading, but is impaired by long-term. We speculated that mitochondrial fission inhibition is associated with the development of NASH and generated mice deletion of hepatocyte-specific mitochondrial fission factor (KO) mice. Conditioning HFD, KO mice showed inhibition of hepatocyte mitochondrial fission, increased ER stress and decreased triglyceride secretion from the liver. The hepatic triglyceride contents had significantly increased in KO mice compared to control (CONT), and histological analysis showed a marked increase in large lipid droplets (LDs). In addition to marked fat accumulation, hepatic apoptosis, inflammation and fibrosis were increased in the livers of KO mice, indicating the development of NASH. We noted that large LDs were increased in KO mice and added an additional analysis to determine how mitochondrial fission inhibition is involved in LDs formation in the liver. Oleic acid is a representative monounsaturated fatty acid that promotes LDs deposition in hepatocytes and is mildly cytotoxic by inducing ER stress and apoptosis. Loading primary cultured hepatocytes (PH) with oleic acid increased the number of smaller LDs in PH of CONT mice, whereas LDs in cells of KO mice became larger. Gene expression of Cidec, which acts on the fusion of LDs, was increased in the liver of KO mice, suggesting that inhibition of mitochondrial fission may induce the fusion of LDs and the enlargement of intracellular LDs in the liver. Further analysis is planned, as inhibition of mitochondrial fission may be involved in changes of LDs morphology.

Disclosure

Y. Takeichi: None. T. Miyazawa: None. Y. Ogawa: None.
278-LB: The Effect of Combining Very-Low-Calorie Diet with Semaglutide on Beta-Cell Function in Individuals with Type 2 Diabetes
Authors: ANYIAM OLUWASEUN , PHILLIPS BETHAN E. , WILKINSON DANIEL J. , SMITH KEN , ATHERTON PHILIP J. , IDRIS ISKANDAR R.
Published: June 2023

Type 2 diabetes (T2D) is characterised by the loss of the first phase response of insulin-producing pancreatic beta cells. Recently, Very-low calorie diets (VLCD - defined as less than 800 kilocalories per day) have been shown to restore this first phase response to induce remission of T2D, while Glucagon-like peptide-1 agonists also improve the first phase response by enhancing postprandial insulin secretion. Here, we present interim results of an investigation exploring the effect of combining these two interventions on beta cell function, in individuals with T2D.

Eighteen individuals with T2D and body mass index >27kg.m-2 were randomly allocated into 3 groups: i) Semaglutide (S), ii) VLCD (V), and iii) Semaglutide-plus-VLCD (SV). Semaglutide was administered once weekly gradually up titrated to a maximum dose of 1mg and VLCD regime was provided by LighterLife® total meal replacement. Beta cell function was assessed using intravenous glucose tolerance tests (IVGTT) performed at baseline and 12 weeks. First-phase insulin response was calculated as the incremental area under the suprabasal plasma insulin concentration curve during 0-10 minutes of the IVGTT (µU.min.ml-1). Data were analysed using GraphPad Prism 9.5.0 (La Jolla, USA).

All groups were equally matched at baseline. Mean weight loss was 6.4, 14.1 and 14.9 kg in S, V and SV, respectively. A significant improvement in first phase response was observed in all groups (S: +398.5; V: +88.4; SV: +547.8 µU.min.ml-1, all p<0.05 vs. baseline). At 12 weeks, first phase response in SV was significantly higher than in V (583.9 vs. 169.2 µU.min.ml-1 respectively, p<0.05).

These data suggest that combination of VLCD and Semaglutide results in a significantly greater improvement in first phase response than VLCD alone. This approach could result in a more robust remission of T2D in the longer-term, and may have important implications for the future design of interventions for the clinical management of T2D.

Disclosure

O. Anyiam: None. B. E. Phillips: None. D. J. Wilkinson: None. K. Smith: None. P. J. Atherton: None. I. R. Idris: None.

Funding

Medical Research Council Confidence in Concept Scheme
278-OR: Impact of a Parameter Change in DBLG1 System
Authors: ADENIS ALICE , VESIN AURÉLIEN , ROMERO-UGALDE HECTOR M. , LOUIS MAXIME , HUBER CLÉMENT V. , POU LIM-SYLVIE , TOURKI YOUSRA , HUNEKER ERIK , BENHAMOU PIERRE Y.
Published: June 2023

In DBLG1 System, multiple parameters can be customized. This study aims at analyzing the impact of a change of the normoglycemia aggressiveness (NA), the hyperglycemia aggressiveness (HA) and the glucose target (GT) on the glycemic control of T1D patients. Data is collected from 5977 adult patients who gave their consent (3/2021 to 11/2022). Periods of 14 days before and after a single parameter change are collected. Conditions of 70% of data availability and of 75% of time spent in closed loop are requested for each period. The time in range 70-180 mg/dL (TIR), the time below range 70 mg/dL (TBR), the mean sensor glucose (MG), the coefficient of variation (CV) and the mean daily insulin dose (MDI) of the two periods are compared for each parameter using a Wilcoxon Signed-Rank test. Control for the multiple tests is done using the Benjamini-Hochberg procedure. The size effect is inferred using Cohen’s d. The results are summarized in the table below. On average, an increase of HA or NA improves the TIR and reduces the MG. The TBR appears to increase when the HA is increased and to decrease when HA or NA are reduced. A decrease of the GT increases TIR, TBR and CV, and decreases MG, conversely an increase of the GT decreases TIR, TBR and CV, and increases MG. The size effect is small. The impact on MDI is small or not significant. The most impactful parameter seems to be the GT which affects the MG proportionally. HA and NA had a lower than expected impact.

Disclosure

A.Adenis: Employee; Diabeloop SA. A.Vesin: Employee; Diabeloop SA. H.M.Romero-ugalde: Employee; Diabeloop SA. M.Louis: Employee; Diabeloop SA. C.V.Huber: Employee; Diabeloop SA. L.Pou: None. Y.Tourki: Employee; Diabeloop SA. E.Huneker: Employee; Diabeloop SA. P.Y.Benhamou: Advisory Panel; Eli Lilly and Company, Insulet Corporation, Novo Nordisk, Consultant; Dexcom, Inc., Employee; Diabeloop SA.
279-OR: Placental IGFBP1 Is Associated with Insulin Sensitivity during Pregnancy and Is an Early Biomarker for Gestational Diabetes
Authors: HIVERT MARIE-FRANCE , WHITE FRÉDÉRIQUE , ALLARD CATHERINE , JAMES KAITLYN , MAJID SANA , FLOREZ JOSE C. , EDLOW ANDREA , BOUCHARD LUIGI , JACQUES PIERRE-ETIENNE , ANANTH KARUMANCHI S. , POWE CAMILLE E.
Published: June 2023

Pregnancy is associated with a profound reduction in insulin sensitivity (IS) that contributes to gestational diabetes (GDM), but the placental factors that lower IS in pregnancy are not fully known. We conducted genome-wide RNA sequencing in 450 placenta samples from a prospective pregnancy cohort (Gen3G). Participants provided plasma samples in the 1st trimester and completed 75g oral glucose tolerance tests in the late 2nd trimester, from which IS was estimated via the Matsuda index. We used regression analyses to test differential expression of 15,232 placental genes related to maternal IS, adjusting for gestational age at delivery, fetal sex, maternal age, gravidity, BMI, and 37 surrogate variables (to account for technical variability). Among the top differentially expressed genes, lower placental expression of IGFBP1 was significantly associated with lower maternal IS (P=1.7×10-4). In 837 Gen3G women, lower IGFPB1 circulating protein was strongly associated with lower IS (r=0.50, P<0.001) and predicted GDM risk (1st trimester median levels: 23,884 pg/mL in no GDM vs 14,363 pg/mL in GDM, adjusted OR=0.55 [0.39-0.75] per quartile increase; P=1.5×10-4). The association between plasma IGFBP1 and IS was replicated in two pregnancy cohorts: SPRING (n=165 with GDM risk factors, r=0.54, P<0.001) and MOMS (n=97 matched GDM cases and controls, r=0.55, P<0.001). The association of lower plasma IGFBP1 with greater risk of GDM was replicated in SPRING (adjusted OR=0.66 per quartile increase, P=0.07) and MOMS (adjusted OR=0.45 per quartile increase, P<0.001). In SPRING, plasma IGFBP1 protein were markedly elevated in the 1st and 2nd trimesters compared to postpartum (P<0.001), consistent with placental origin. Our results suggest that the placenta expresses high levels of IGFBP1, which influences IGF-1 bioavailability and is released in maternal circulation to regulate IS in pregnancy, highlighting it as a promising early plasma biomarker of GDM risk.

Disclosure

M.Hivert: None. S.Karumanchi: None. C.E.Powe: Consultant; Mediflix, Inc., Other Relationship; Wolters Kluwer Health. F.White: None. C.Allard: None. K.James: None. S.Majid: None. J.C.Florez: Consultant; AstraZeneca, Novo Nordisk, Other Relationship; AstraZeneca, Merck & Co., Inc. A.Edlow: Consultant; Mirvie, Inc, Research Support; Merck & Co., Inc. L.Bouchard: None. P.Jacques: None.

Funding

Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD094150)