bluuebunny/crossref_metadata_2025 huggingface dataset:
1701-P: The Role of UGT1a in Regulating Brown Adipose Tissue and Thermogenic Function
Authors: KODANI SEAN , LYNES MATTHEW , SHAMSI FARNAZ , WANG CHIH-HAO , SUGIMOTO SATORU , XUE RUIDAN , HUANG TIAN LIAN , WHITE ANDREW P. , CYPESS AARON M. , TSENG YU-HUA
Published: June 2020

Increasing biological thermogenesis (the conversion of chemical energy to heat) has been proposed as an approach towards lowering blood glucose, improving insulin sensitivity and mitigating type 2 diabetes. Non-shivering thermogenesis occurs primarily in adipocytes that express uncoupling protein 1 (UCP1) including brown adipose tissue (BAT) and beige adipose tissue but does not substantially occur in white adipose tissue (WAT). To identify genes that regulate thermogenesis and BAT function, we recently compared genes expressed in paired human patient samples (n = 10) of the deep neck BAT located next to the carotid sheath and superficial subcutaneous WAT in the neck. We found UDP-glucuronide transferase 1A (UGT1A) is co-expressed in individuals with a high amount of the thermogenic protein uncoupling protein 1 (UCP1). This enzyme is known to glucuronidate metabolites, including bilirubin and xenobiotics, which increases their water solubility and excretion. Based on this function, we hypothesize UGT1A regulates BAT not through direct action of the enzyme but through modulating the availability of thermogenic molecules. Here, we characterized expression of different UGT1A isoforms in vivo and in vitro. Cold exposure is an effective method for activating BAT and increasing thermogenesis in mice. Interestingly, expression of multiple UGT1A isoforms, including UGT1A6a and UGT1A6b, are decreased in BAT of mice exposed to a cold environment (5°C) for 7 days. This finding is consistent with data in multiple publicly available datasets which have shown UGT1A6a/6b are decreased in cold exposure and other thermogenic stimuli. In vitro, most UGT1A isoforms were increased during the differentiation of murine brown adipocytes but only certain isoforms were differentially expressed during in vitro differentiation in human brown adipocytes. Based on these data, we propose that UGT1A may have a function related to thermogenesis and understanding its function could lead to novel approaches for activating BAT.

Disclosure

S. Kodani: None. M. Lynes: None. F. Shamsi: None. C. Wang: None. S. Sugimoto: None. R. Xue: None. T. Huang: None. A.P. White: None. A.M. Cypess: None. Y. Tseng: Consultant; Self; Boehringer Ingelheim International GmbH.

Funding

National Institutes of Health (R01DK07709, R01DK102898, T32DK007260)
1712-P: HIV-1 Vpr Couples Metabolic Inflexibility with White Adipose Tissue Thermogenesis
Authors: AGARWAL NEETI , SAHA PRADIP , HARTIG SEAN M. , BALASUBRAMANYAM ASHOK
Published: June 2020

The HIV accessory protein Vpr contributes to cardinal metabolic defects noted in persons living with HIV (PLWH), including accelerated lipolysis, impaired fatty acid oxidation, slowed fatty acid export, and steatohepatitis (Agarwal et al, Sci Trans Med 2017). PLWH also manifest “beiging” and markers of thermogenesis within some white adipose tissue (WAT) depots, but paradoxically these changes correlate with decreased insulin sensitivity and hyperlipidemia. We sought to determine whether Vpr activity affects thermogenesis and the dynamics of beiging in two Vpr mouse models. UCP1 expression was strongly increased in subcutaneous fat of Vpr mice and showed equivalent copy number in WAT biopsies from PLWH. Histology confirmed browning of subcutaneous but not visceral WAT in the Vpr mouse models. Adipocytes derived from these depots showed increased oxygen consumption rate in subcutaneous but not visceral fat cells, also strongly indicative of beiging. Measurements of energy balance indicated Vpr mice display metabolic inflexibility and cannot shift efficiently from carbohydrates to fat during day-night cycles. Furthermore, Vpr mice mice showed a marked inability to defend body temperature when exposed to 4oC for 6 hours. Thus HIV-1 Vpr disrupts metabolic efficiency coupled with maladaptive subcutaneous WAT beiging. These data suggest an expanded role for Vpr in the pathogenesis of HIV-associated metabolic disorders.

Disclosure

N. Agarwal: None. P. Saha: None. S.M. Hartig: None. A. Balasubramanyam: None.

Funding

National Institutes of Health (R21/R33AI116208)
1713-P: Adipocyte O-GlcNAc Transferase Regulate Adipogenesis through De Novo Lipogenesis in Mice
Authors: MORINO KATSUTARO , TSUJI AKIKO , OHASHI NATSUKO , IDA SHOGO , PERRY RACHEL J. , UGI SATOSHI , FUJITA YUKIHIRO , SHULMAN GERALD I. , MAEGAWA HIROSHI
Published: June 2020

O-linked N-acetylglucosamine (O-GlcNAc) modification is one of the post-translational modifications which modulates protein fate and function, and it is mediated by O-GlcNAc transferase (OGT). The substrate of OGT, UDP-GlcNAc, is biosynthesized through the branch of glycolysis. Thus O-GlcNAcylation has been regarded as a nutrient sensor. Metabolic regulation in white adipose tissues (WAT) is dramatically changed in the process of obesity. We previously reported that the deletion of Ogt in adipose tissues (Ogt-FKO) decrease WAT mass without affecting glucose tolerance in mice under normal condition. However, the role of O-GlcNAcylation in WAT in the process of being obesity remains unknown. In this study, we investigated the role of O-GlcNAcylation in WAT under diet induced obesity. Despite no significant differences in food intake, body weight gain by high-fat diet was suppressed in Ogt-FKO mice along with the significant decrease of WAT mass. However, Ogt-FKO mice were severely impaired glucose tolerance and decreased insulin sensitivity in liver, which was confirmed by hyper-insulinemic euglycemic clamp. Histologically, we observed smaller adipocytes and fibrosis in WAT of Ogt-FKO mice. In addition, gene expression of F4/80, CD11c, MCP-1 and Col1a1 were significantly higher in Ogt-FKO mice, suggesting increased inflammation and fibrosis. Also, the expression of genes associated to fatty acid synthesis such as SREBP-1c, FAS, SCD1 and SCD2 were reduced in Ogt-FKO mice. Furthermore, Ogt-FKO mice showed lower serum concentration of adiponectin and leptin (46.43 ± 3.62 vs. 8.42 ± 1.53ng/ml) along with their gene expression, which is consistent with severe atrophy of WAT in Ogt-FKO mice.

In conclusion, loss of O-GlcNAcylation in WAT exhibited obesity resistance and impaired glucose tolerance because of lipoatrophy under diet induced obesity, and the results suggest that O-GlcNAcylation in WAT plays important role for fatty acid synthesis and functional adipocyte formation.

Disclosure

K. Morino: None. A. Tsuji: None. N. Ohashi: None. S. Ida: None. R.J. Perry: Research Support; Self; AstraZeneca. S. Ugi: None. Y. Fujita: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, LLC, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Consultant; Self; Novo Nordisk A/S. Consultant; Spouse/Partner; Novo Nordisk A/S. Other Relationship; Self; Gilead Sciences, Inc., iMetabolic Biopharma Corporation. Other Relationship; Spouse/Partner; iMetabolic Biopharma Corporation. Other Relationship; Self; Maze Therapeutics. H. Maegawa: Speaker’s Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Sanofi K.K., Takeda Pharmaceutical Company Limited.

Funding

Japan Society for the Promotion of Science (18K16226)
1714-P: Regulation of Phosphorylation Status in Insulin Signaling via a4 Is Necessary for Adipocyte Maintenance and Lipid Dynamics
Authors: SAKAGUCHI MASAJI , OKAGAWA SHOTA , OKUBO YUMA , KITANO SAYAKA , IGATA MOTOYUKI , KONDO TATSUYA , ARAKI EIICHI
Published: June 2020

Insulin and IGF1 signaling through the PI3K/AKT and MAPK/ERK pathways are essential for maintenance of brown and white adipose tissues (BAT and WAT), as shown by our previous results using adipocyte-specific and inducible IR/IGF1R knockout mice (Ai-DKO). Here, through a research to screen the genes essential for adipose tissues maintenance in WAT and BAT using Ai-DKO mice, we identified a reduction of α4, a protein phosphatase protector which regulate phosphorylation state, in Ai-DKO BAT. shRNA-mediated α4 knockdown (KD) altered insulin-stimulated phosphorylation status in BAT; decreased IRβ (Y1162/1163), IRS1 (Y612) and Akt (S473), with a mild change in ERK1/2 (T202/Y204) but increased ribosomal S6 protein (S235/236), suggesting α4 reduces S6 phosphorylation state via a unique pathway. Next, to investigate the impact of α4 in vivo, we created inducible adipocyte-specific α4 KO (Ai-α4 KO) mice with tamoxifen-inducible Cre-ERT2 transgene. Once induced α4 KO in adipocytes, the mice revealed extensive losses of adipocytes in SC-WAT and BAT depots with increased apoptotic cell death. Ai-α4 KO mice showed cold intolerance, severe diabetes, ectopic lipid accumulation in the liver, and pancreatic islet hyperplasia. RNA-seq showed a marked reduction of genes associated with mitochondrial fatty acid oxidation and increases with inflammatory cytokine pathways in Ai-α4 KO SC-WAT and BAT. WAT and BAT mass can recover after a few months in Ai-α4KO. Interestingly, lipidomics analysis displayed that the regenerated adipocytes showed unique lipid content in the Ai-α4KO. Thus, our model demonstrates that the tuning of phosphorylation status for insulin-dependent signaling by α4 is critical for maintaining and regeneration of white and brown adipose tissues.

Disclosure

M. Sakaguchi: None. S. Okagawa: None. Y. Okubo: None. S. Kitano: None. M. Igata: None. T. Kondo: None. E. Araki: Advisory Panel; Self; Abbott. Speaker’s Bureau; Self; ARKRAY, Astellas Pharma Inc., AstraZeneca, Eli Lilly Japan K.K., Merck & Co., Inc., Novo Nordisk Inc., WebMD LLC. Other Relationship; Self; Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation, Novartis Pharma K.K., Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited.

Funding

Japan Society for the Promotion of Science; Merck Sharp & Dohme Foundation; Takeda Foundation; Boehringer Ingelheim; Eli Lilly and Company
1716-P: L-PGDS Inhibition Reduces Adiponectin Levels and Insulin Sensitivity in 3T3-L1 Adipocytes
Authors: SRIVASTAVA ANKITA , PALAIA THOMAS , LEE JENNY , HALL CHRIS , STEVENSON MATTHEW , FERNANDO ANNE G. , RAGOLIA LOUIS
Published: June 2020

The prevalence of obesity has increased steadily over the last few decades and is a major risk factor for type 2 diabetes and cardiovascular disease. Obesity is associated with alterations in adipose tissue functioning leading to metabolic dysfunction. In previous studies, we have reported the role of lipocalin-type prostaglandin D2 synthase (L-PGDS) in glucose intolerance and atherosclerosis. More recently, we demonstrated that L-PGDS KO mice develop NAFLD even on a low fat diet. Our present study explored the role of L-PGDS in adipose tissue. We found decreased expression of L-PGDS in insulin resistant 3T3-L1 adipocytes. Enzymatic inhibition of L-PGDS in 3T3-L1 adipocytes using AT56 impaired insulin signaling by reducing the phosphorylation of AKT protein and the expression and translocation of GLUT4 to plasma membrane. Furthermore, we found that inhibition of L-PGDS reduces the expression of adiponectin and AMPK phosphorylation in 3T3-L1 adipocytes. Rosiglitazone treatment in insulin resistant 3T3-L1 adipocytes significantly increased L-PGDS expression. Treatment with rosiglitazone restored the expression of adiponectin and phosphorylation of AMPK in AT56 treated 3T3-L1 adipocytes. Correspondingly, we found decreased L-PGDS expression in visceral fat of obese humans. Cumulatively, these data confirm that the absence of L-PGDS results in decreased insulin sensitivity via impaired insulin signaling and reduced adiponectin levels in 3T3-L1 adipocytes. Thus, our findings suggest that L-PGDS plays a significant role in maintaining the insulin sensitivity in 3T3-L1 adipocytes and provides a novel opportunity for therapeutic intervention of obesity-associated metabolic syndrome.

Disclosure

A. Srivastava: None. T. Palaia: None. J. Lee: None. C. Hall: None. M. Stevenson: None. A.G. Fernando: None. L. Ragolia: None.

Funding

American Heart Association (15GRNT22420001); George Link, Jr. Foundation, Inc.
1725-P: Viral Insulins as Agonists and Antagonists on Insulin/IGF-1 Receptors
Authors: MOREAU FRANCOIS , BRANDAO BRUNA ,   ESQ., CEDERQUIST CARLY , BATISTA THIAGO M. , DIMARCHI RICHARD , ALTINDIS EMRAH , KAHN C. RONALD
Published: June 2020

Recently, we identified four insulin/IGF-1 like peptides (VILPs) in the sequence of viruses belonging to the Iridoviridae family. These VILPs show up to 50% homology with the sequence of the human insulin/IGF-1. Moreover, the predicted tertiary structures and major receptor binding residues are conserved between the VILPs and insulin/IGF-1. Although these viruses are known to infect primarily fish, sequences of VILPs have been found in human blood and feces. VILPs can interact with the human and murine insulin and IGF-1 receptors and induce the activation of their downstream signaling. Functionally, VILPs are able to induce glucose uptake and cell proliferation.

VILPs can be synthesized as a single-chain (SC) or double-chain (DC) peptides, i.e., more IGF-1 like or more insulin like structures. In the present study, we show that SC and DC forms of lymphocystis disease virus (LCDV-1) VILP interact differently with the human insulin and IGF-1 receptor. Competition binding assay reveals that SC is more potent than DC in binding to the IGF-1 receptor. In vivo, following i.p. injection in mice both LCDV-1 SC and DC produced similar mild decreases in blood glucose compared to insulin, even at a 166-fold excess. In vitro, however, DC LCDV1-VILP acts as a weak agonist to stimulation of IGF1R, whereas SC LCDV-VILP serves as a strong antagonism effect on the IGF-1 receptor. Thus, the activation of the IGF1R by IGF-1 is strongly repressed by the SC LCDV-1 VILP, even at equal concentrations. This antagonistic action of SC VILP is restricted to the IGF1R and does not affect insulin receptor. As a functional consequence of IGF1R blockade, SC LCDV-1 VILP inhibits IGF-1 stimulated migration of a cervical cancer cell line.

In summary, we have shown that SC VILP may provide a novel inhibitor of IGF1R signaling and IGF-1 action on tumor cell migration, with minimal effects on glucose metabolism, thus providing a potentially unique reagent for treatment of IGF-1 sensitive tumors.

Disclosure

F. Moreau: None. B. Brandao: None. C. Cederquist: None. T.M. Batista: None. R. DiMarchi: Employee; Self; Novo Nordisk Inc. E. Altindis: None. C. Kahn: Advisory Panel; Self; ERX Pharmaceuticals, Kaleido Biosciences. Consultant; Self; AntriaBio, Flagship Pioneering, Sana-Cobalt.
1727-P: Mice Deficient in Myeloid Cell-Derived Interleukin-1a Are Protected from Diet-Induced Obesity and Insulin Resistance
Authors: SUK SUJIN , FRIEDLINE RANDALL H. , NOH HYE LIM , HU XIAODI , TRAN DUY A. , TAUER LAUREN A. , KIM ALLISON M. , KIM LAUREN H. , KIM JASON K.
Published: June 2020

Interleukin (IL)-1α is a major mediator of inflammation, and functional polymorphism of IL-1α is associated with human obesity. We have recently generated mice with genetic ablation of IL-1α selectively in myeloid cells. On chow diet, Lyz-IL-1α KO mice showed normal metabolic phenotypes. In contrast, when male Lyz-IL-1α KO mice were fed a high-fat diet (HFD), whole body fat mass was markedly reduced in these mice (Figure 1; *P<0.05). Metabolic cage study found that this was mostly due to a 2-fold increase in physical activity in Lyz-IL-1α KO mice (Figure 2). Food intake and energy expenditure were not affected in these mice. We performed a hyperinsulinemic-euglycemic clamp to measure insulin sensitivity in awake mice (n=7). HFD-fed Lyz-IL-1α KO mice were more insulin sensitive than HFD-fed wild type (WT) mice with a 2-fold increase in glucose infusion rates (Figure 3). Whole body glucose turnover and glycolysis were significantly increased in HFD-fed Lyz-IL-1α KO mice (Figure 3). Basal hepatic glucose production (HGP) was not altered, but clamp HGP was reduced by 40% in Lyz-IL-1α KO mice. As a result, hepatic insulin action was markedly increased in HFD-fed Lyz-IL-1α KO mice (Figure 4).

In conclusion, these results indicate that Lyz-IL-1α KO mice are protected from diet-induced obesity and insulin resistance, and our findings identify IL-1α as a novel therapeutic target in obesity and type 2 diabetes.

Disclosure

S. Suk: None. R.H. Friedline: None. H. Noh: None. X. Hu: None. D.A. Tran: None. L.A. Tauer: None. A.M. Kim: None. L.H. Kim: None. J.K. Kim: None.

Funding

National Institutes of Health (5U2CDK093000)
1728-P: Oral Consumption of a PI3K P110a Inhibitor Perturbs Glucose Homeostasis without Affecting the Physical or Cognitive Function of Adult Mice
Authors: HEDGES CHRISTOPHER , BOIX JORDI , MERRY TROY L.
Published: June 2020

Advances in medicine have contributed to an ageing population, and various age-associated pathologies contribute to loss of independence and quality of life. Calorie restriction and genetic inhibition of insulin signalling pathway intermediates such as phosphatidylinositol-3-kinase (PI3K), have shown promise in improving lifespan but are difficult to implement clinically. Therefore, we sought to test whether pharmacological inhibition of PI3K using an orally ingested p110α-selective inhibitor, BYL-719, promotes changes in glucose homeostasis and physical function in adult (8-month old) male and female mice. Three days of exposure to a standard chow diet containing 0.30 g/kg BYL-719 elevated blood glucose in response to a meal challenge, however, following four weeks of BYL-719 diet consumption blood glucose response to a meal was normalised in females, but not males. Despite this, both male and female mice fed the BYL-719 diet were glucose intolerance and had impaired insulin sensitivity compared to mice fed a control diet. There was no difference between groups in measures of physical performance (treadmill, rotarod or hang time performance) or stress/anxiety behaviour assessed through open field test. However, female mice fed the BYL-719 diet showed greater exploratory behaviour in an object recognition test. Collectively, these data indicate that short-term pharmacological inhibition of PI3K p110α alters glucose homeostasis, but has only mild effect with respect to the physiological stimulus of a meal, and without deterioration of physical or cognitive function in adult mice. This indicates BYL-719 is safely tolerated in adult mice, and longer-term study is warranted to test the efficacy of BYL-719 in prolonging lifelong health in an aged population.

Disclosure

C. Hedges: None. J. Boix: None. T.L. Merry: None.

Funding

Health Research Council of New Zealand
1738-P: The Role of Adipocyte YAP in Glucose Homeostasis and Insulin Resistance In Vivo
Authors: HAN DANIEL JONG JIN , ASLAM RUKHSANA , OJHA TANVI , YUEN DARREN , LUK CYNTHIA T.
Published: June 2020

Adipose tissue is a dynamic metabolic organ that is essential for the regulation of whole-body glucose homeostasis. Adipose tissue hypertrophy with obesity is associated with glucose intolerance and insulin resistance underlying type 2 diabetes. Furthermore, studies suggest fibrosis formation is increased in adipose tissue of mice fed with a high-fat diet (HFD) and results in metabolically dysfunctional adipose tissue. Yes-associated protein 1 (YAP) is a transcription cofactor that promotes fibrosis in the lung, liver, and kidney. However, the role of YAP in adipose tissue and glucose homeostasis is unknown. To understand the role of YAP in vivo under metabolic stress conditions, we assessed how HFD impacts YAP protein levels. We have found that YAP protein levels increase in adipose tissue with weight gain. This suggests that YAP signaling may contribute to adipocyte dysfunction and insulin resistance under metabolic stress conditions. To further investigate the role of YAP in adipose tissue and glucose homeostasis in vivo, we developed adipose tissue-specific YAP knockout (adipose tissue YAP-KO) mice and placed them on both chow and HFD for 12 weeks. On an HFD, adipose tissue YAP-KO mice show improved glucose tolerance compared to controls. While blood glucose levels were lower, adipose tissue YAP-KO mice did not show improvements in insulin sensitivity. Together, these data indicate that YAP increases in adipose tissue with weight gain and disruption of YAP in adipocytes improves glucose tolerance; suggesting that adipocyte YAP plays an important role in modulating glucose homeostasis under metabolic stress conditions.

Disclosure

D. Han: None. R. Aslam: None. T. Ojha: None. D. Yuen: None. C.T. Luk: None.
1746-P: Obese Youth with Type 1 Diabetes (T1D) Have Worse Hepatic Insulin Resistance (IR) Than Youth with Type 2 Diabetes (T2D)
Authors: WIROMRAT PATTARA , CREE-GREEN MELANIE , BERGMAN BRYAN C. , TOMMERDAHL KALIE L. , BAUMGARTNER AMY , PYLE LAURA , NADEAU KRISTEN J.
Published: June 2020

Background: The epidemic of obesity is growing in the U.S. due to changes in diet and lifestyle. Similar trends are also now occurring in T1D youth and may worsen IR and associated cardiovascular risk. However, the impact of obesity on IR in each tissue is unknown in T1D. Thus, we evaluated adipose, hepatic and muscle IR in obese youth with T1D, compared to BMI-similar control or T2D youth.

Methods: 20 youth with T1D (age 16.0±2.3 years; % female 55; BMI%ile 95th [97, 98]; HbA1c 8.7±1.6%), 20 youth with T2D (age 15.4±2.3; % female 52; BMI%ile 95th [98, 99]; HbA1c 8.5±2.4%) and 16 controls (age 14.8±1.8; % female 55; BMI%ile 97th [98, 99]) were included. In those with diabetes, glucose was controlled overnight by IV insulin to a target of ∼100 mg/dL. A 4-phase hyperinsulinemic-euglycemic clamp (basal, 10, 16 and 80 mU/m2/min of insulin [I]) with isotopic tracers was performed the following AM to assess adipose, hepatic and muscle IR, respectively. Free fatty acids (FFA), glucose rate of disappearance (Rd) and glycerol and glucose rate of appearance (Ra) and %suppression (basal to 80 phase) were calculated.

Results: Participant age, sex, BMI and Tanner stage were similar between groups. HbA1c was similar in groups with diabetes. During the basal phase, glycerol Ra/I and glucose Ra/I were similar between groups, but FFA/I was highest in obese controls (p<0.001). At phase 10, glycerol Ra/I was similar between groups but FFA/I at phase 10 and 16 were similarly higher in T1D and T2D vs. controls (p=0.004, p<0.001). At phase 16, glucose Ra/I and glycerol Ra/I were highest in T1D (p<0.001, p<0.001). At phase 80, T1D vs. T2D had similarly lower %glycerol Ra suppression than controls (p<0.001) but higher glycerol Ra/I and glucose Rd in T1D vs. T2D (p=0.003, p=0.042). T1D youth and controls had similar glucose Rd (p=0.25).

Conclusion: Obese youth with T1D have similar to worse adipose, worse hepatic, and less muscle IR than those with T2D. Treatment targeting obesity and IR is critically needed in T1D youth.

Disclosure

P. Wiromrat: None. M. Cree-Green: Advisory Panel; Self; Novo Nordisk Inc. B.C. Bergman: Consultant; Spouse/Partner; Novo Nordisk Inc., Sanofi US. Research Support; Self; Eli Lilly and Company. Speaker’s Bureau; Spouse/Partner; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc. K.L. Tommerdahl: None. A. Baumgartner: None. L. Pyle: None. K.J. Nadeau: None.

Funding

American Diabetes Association (7-11-CD-08 to K.J.N.); National Heart, Lung, and Blood Institute; National Institute of Diabetes and Digestive and Kidney Diseases; JDRF
1748-P: One Novel 7.2kb Deletion in Exon 8 of INSR Gene in Diabetic Qatari Female with Type A Insulin Resistance Syndrome: The 1000 Qatar-Omics Study Cohort
Authors: AKIL AMMIRA S. , YASSIN ESRAA A.M. , PADMAJEYA SUJITHA SUBASH , JERMAN LAILA A. , FADDA ABEER , AAMER WALEED H.O. , ALIYEV ELBAY E. , FAKHRO KHALID
Published: June 2020

Background: A 46-year-old Qatari female with a BMI of 25kg/m2 presented with hyperglycemia (blood glucose 13.5 mmol/l), hyperinsulinemia (21µU/mL) with elevated levels of HbA1c (10.3mmol/l). Biochemical investigations showed raised serum C-peptide (2.02ng/ml) and triglycerides (2.07mmol/L) and uric acid (104mg/dL), with normal BP. The objective of this case study is to validate this variant found through WGS in insulin receptor (INSR) gene that play a major cause of insulin resistant syndrome with different spectrums.

Methods: Structural Variants discovered using custom multi-algorithm pipeline developed and optimized to run on HPC for short-read WGS data. In brief, 10 software packages applied (Breakdancer, Breakseq2, CNVnator, Delly, ERDS, Genomestrip, Manta, Speedseq, Svaba, WHAM), to existing short-read data. SVs annotated using AnnotSV and provides us functionally, regulatory and clinically relevant information. SVs analyzed based on their properties like pli-score, overlaps with known databases and OMIM annotations. This deletion was validated by ddPCR and the molecular docking performed by the autodock suit in order to understand the effect of deletion in the interaction between this receptor and insulin.

Results: The patient was identified with 7.2k heterozygous deletion in exon 8 of INSR gene which play an essential role in the formation of extracellular alpha subunits in insulin receptor. molecular docking proved the decrease of catalytic activity of INSR and, hence signaling. Considering this and the phenotypic data from Qatar biobank, the patient developed features of type A insulin resistant syndrome as consequence of persistence hyperinsulinemia. The patient also exhibits severe Deterioration of glucose tolerant even with medication and unfortunately developed nephropathy.

Conclusion: We highlighted the power of NGS in precise diagnosis and developing a potential and promising targeted therapeutics.

Disclosure

A.S. Akil: None. E.A.M. Yassin: None. S. Subash Padmajeya: None. L.A. Jerman: None. A. Fadda: None. W.H.O. Aamer: None. E.E. Aliyev: None. K. Fakhro: None.
175-OR: Impact of High-Deductible Health Plan Enrollment on the Timing of Diabetic Retinopathy Care and Vision Loss Diagnosis
Authors: WHARAM J. FRANK , ZHANG FANG , LU CHRISTINE , STRYJEWSKI TOMASZ , CALLAWAY KATHERINE , WALLACE JAMIE , NEWHOUSE JOSEPH P. , ROSS-DEGNAN DENNIS
Published: June 2020

High-deductible health plans (HDHP) that require large out-of-pocket payments for retinopathy screening and ophthalmologist visits could delay retinopathy screening, retinopathy diagnosis, and vision loss diagnosis. We studied 2004-2014 data in a large commercial and Medicare Advantage claims database. We included a national sample of 39,798 HDHP members with diabetes enrolled for 1 year in a low deductible (≤$500) plan followed by up to 4 years in a HDHP (≥$1000) after an employer-mandated switch. We matched HDHP patients to 305,852 contemporaneous controls whose employers offered only low-deductible plans. Outcomes included time to first: (a) retinopathy screening, (b) diagnosis of mild-to-moderate and (c) severe non-proliferative diabetic retinopathy, and (d) vision loss diagnosis. We used Cox proportional hazards models adjusted for multiple baseline characteristics to estimate baseline and follow-up hazard ratios. At baseline, adjusted hazard ratios (aHRs) between HDHP members and controls were not statistically different. At follow up, HDHP members experienced delays in first retinopathy screening (aHR: 0.89 [085, 0.92]), diagnosis of mild-to-moderate non-proliferative diabetic retinopathy (aHR: 0.82 [0.76, 0.89]), diagnosis of severe non-proliferative diabetic retinopathy (aHR: 0.90 [0.81, 1.00]), and vision loss diagnosis (aHR: 0.93 [0.88, 0.99]). HDHP members with diabetes experienced delayed retinopathy screening, retinopathy diagnosis, and vision loss diagnosis compared with similar patients in more generous health plans. Reducing financial barriers to care might improve retinopathy outcomes.

Disclosure

J. Wharam: None. F. Zhang: None. C. Lu: None. T. Stryjewski: Other Relationship; Self; Aldeyra Therapeutics, Pykus Therapeutics. K. Callaway: None. J. Wallace: None. J.P. Newhouse: None. D. Ross-Degnan: None.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases
1750-P: Effects of Melatonin on the Insulin Signaling and Inflammatory Pathway of Rats with Apical Periodontitis
Authors: SUMIDA DORIS H.
Published: June 2020

Background: Apical periodontitis (PEA) is oral inflammation related to the increase of proinflammatory cytokines that may act locally and systemically. PEA in rats has been shown to cause insulin resistance (IR) and impaired insulin signal. Melatonin (MEL) is known to improve IR. We hypothesized that administration of MEL in PEA rats may prevent the IR found in these animals. Therefore, the aim of this study was to verify, in rats with PEA, the effects of MEL on: IR; the insulin and inflammatory pathways; TNF-α levels.

Methodology: Seventy-two Wistar rats (60 days of age) were distributed into: control (CN); CN+MEL (CNMEL); PEA; PEA+MEL (PEAMEL). PEAs were induced in the first and second upper and lower molars of the right side using a surgical drill. Following PEA induction, MEL (5 mg/kg) was administered orally (drinking water) for 60 days. At the end of treatment were analyzed: 1) HOMA-IR; 2) TNF-α levels; 3) phosphorylation status of pp185 tyrosine, Akt serine and IKKα/β in soleus (SM) and extensor digitorum longus (EDL) muscles. Statistical analysis was performed by analysis of variance (ANOVA) followed by Bonferroni test (p<0.05).

Results: HOMA-IR and TNF-α were higher in the PEA group than in the other groups (HOMA-IR: CN: 2.49 ± 0.24; PEA: 3.57 ± 0.24; CNMEL: 2.17 ± 0.13; PEAMEL: 2.57 ± 0.09 - TNF-α: CN: 8.56 ± 0.84; PEA: 13.42 ± 0.86; CNMEL: 9.21 ± 0.83; PEAMEL: 8.62 ± 0.42). The phosphorylation status of pp185 tyrosine and Akt serine was lower in the PEA group compared to the CN group in SM. Administration of MEL in the PEAMEL group increased the phosphorylation of both proteins. No statistical differences in these parameters were observed between the groups in the EDL muscle. Higher IKKα/β phosphorylation was observed in the PEA group compared to the other groups in both tissues.

Conclusions: PEA promoted IR, increased TNF-α levels, impaired insulin signal in SM and inflammatory pathway activation in SM and EDL. Administration of MEL only prevented changes in insulin signal in MS.

Disclosure

D.H. Sumida: None.

Funding

Fundação de Amparo a Pesquisa do Estado de São Paulo (2018/23346-3, 2019/18589-7); São Paulo State University
1753-P: Insulin Sensitivity in Long-Lived Growth Hormone–Releasing Hormone Knockout Mice
Authors: ZHANG FANG , ICYUZ MERT , SUN LIOU
Published: June 2020

Disruption of growth hormone releasing hormone results in extended longevity with increased insulin sensitivity in mice. Yet, less is known about tissue-specific insulin action and insulin signaling pathway in growth hormone releasing hormone knock-out mice (GHRH-/-). In this study, our hyperinsulinemic-euglycemic clamp data showed that glucose infusion rate to maintain euglycemia was dramatically elevated in GHRH-/- mice. Insulin-mediated hepatic glucose production was suppressed whereas glucose uptake in skeletal muscle and brown adipose tissue were significantly increased in GHRH-/- mice compared to WT littermates. Furthermore, growth hormone deficiency further enhanced insulin sensitivity in metabolically active tissues via activated PI3K-AKT and MAPK-ERK1/2 signaling cascades. Altered tissue-specific insulin response is likely to mediate the prolonged longevity and healthy-aging effects of growth hormone deficiency in mice.

Disclosure

F. Zhang: None. M. Icyuz: None. L. Sun: None.

Funding

National Institutes of Health (R01AG057734)
1755-P: Insulin-Mediated Glucose Disposal and Muscle Microvascular Recruitment Are Mutual Predictors in Humans
Authors: LOVE KAITLIN , JAHN LINDA , HARTLINE LEE , BARRETT EUGENE , LIU ZHENQI
Published: June 2020

Insulin recruits muscle microvasculature to enhance insulin and nutrient delivery. Insulin resistance (IR) blunts this microvascular action. While metabolic and microvascular IR usually coexist, and phenotypic traits which foretell metabolic IR are well established, those which predict microvascular IR are unknown. To test whether metabolic IR and its associated traits predict microvascular IR, we performed euglycemic insulin clamps (1 mU/kg/min) in healthy lean (n= 57), healthy obese (n=15) and lean type 1 diabetes subjects (T1D) (n=25), and tested parametric correlations and a multivariate model ANOVA. We measured insulin-mediated whole body glucose disposal (M-value) and insulin-induced changes in muscle microvascular blood volume (ΔMBV) by contrast-enhanced ultrasound. Predictor variables included body mass index (BMI), total body weight (WT), percent body fat (BF), lean body mass, blood pressure, maximal rate of oxygen consumption (VO2max), and plasma levels of LDL cholesterol (LDL-C), HDL cholesterol, triglycerides (TG), fasting insulin (INS) and free fatty acids.

A total of 97 subjects were included (mean age 25.4±6.8, 47% women) in the analyses. Among all factors, only M-value (r=0.23, p=0.023) and VO2max (r=0.20, p=0.047) correlated with ΔMBV. Conversely, INS (r=-0.59, P≤0.001), BF (r=-0.54, p≤0.001), VO2max (r=0.5, p≤0.001), BMI (r=-0.40, p<0.001), WT (r=-0.33, p=0.001), LDL-C (r=-0.26, p=0.009), TG (r=-0.25, p=0.012), as well as ΔMBV correlated with M-value. While both ΔMBV (p=0.045) and TG (p=0.037) independently predicted M-value, only M-value independently predicted ΔMBV (p=0.045).

In conclusion, both M-value and VO2max correlated with but only M-value independently predicted microvascular IR. This suggests that, while metabolic and microvascular IR are important predictors of one another, most phenotypic predictors of metabolic IR do not predict microvascular IR.

Disclosure

K. Love: None. L. Jahn: None. L. Hartline: None. E. Barrett: None. Z. Liu: None.

Funding

American Diabetes Association (1-17-ICST-059 to Z.L.); National Institutes of Health (1F32DK121431-01)
1757-P: Tyrosine Phosphorylation of Phospholipase C in Human Skeletal Muscle Insulin Resistance
Authors: ZHANG XIANGMIN , MESTAREEHI AKTHAM H. , SEYOUM BERHANE , YI ZHENGPING
Published: June 2020

Insulin resistance in skeletal muscle is among the primary contributors to the development of type 2 diabetes (T2D), a leading cause of heart disease, stroke, etc. Over 26 million U.S. adults have T2D. Molecular mechanisms for skeletal muscle insulin resistance remain elusive, limiting strategies to prevent/treat T2D. Phospholipase C, gamma 1 (PLCG1, aka PLC-γ1) serves as a signal transducer of tyrosine kinases and regulates various downstream signaling pathways in vitro. PLCG1 has been shown to interact with insulin receptor (IR) via its pleckstrin homology domain and plays a significant role in insulin-stimulated glucose uptake in adipocytes. Moreover, phosphorylation of tyrosine residue 771 (pTyr771) of PLCG1 has been shown to regulate PLCG1 activity. However, no prior publication shows that PLCG1 pTyr771 is linked with insulin resistance and T2D. Previously, we observed that insulin-stimulated PLCG1 pTyr771 is impaired in skeletal muscle from subjects with T2D compared to lean controls. In the present work, we mutated Tyr771 of PLCG1 to phenylalanine, and observed reduced insulin-stimulated AKT and AS160 phosphorylation in primary human skeletal muscle cells derived from an insulin sensitive participant. To identify the upstream regulator of PLCG1, we carried out in vitro kinase assay by incubating PLCG1 with insulin receptor. PLCG1 was expressed and purified from E. coli. The insulin receptor (active cytoplasmic terminus) was expressed and purified from insect cells. After incubating PLCG1 with insulin receptor in vitro for 30 min in the presence of ATP, PLCG1 phosphorylation was determined by HPLC-ESI-MS/MS. The result demonstrated that insulin receptor phosphorylated Tyr771 of PLCG1. Western blotting using pTyr771 specific antibodies confirmed these findings.

In summary, these results suggest that reduced insulin-stimulated pTyr771 may contribute to the development of insulin resistance and T2D, and the insulin receptor is an upstream regulator of PLCG1 pTyr771.

Disclosure

X. Zhang: None. A.H. Mestareehi: None. B. Seyoum: None. Z. Yi: None.

Funding

American Diabetes Association (1-13-TS-27 to Z.Y.); National Institutes of Health (R01DK107666, R01DK081750 to Z.Y.), (T32HL120822 to A.H.M.); DOTS Scholars (to A.H.M., Z.Y.)
1758-P: Type 2 Diabetes–Associated Mood Disorders: Role of Insulin Resistance in Serotonergic Neurons
Authors: MARTIN HUGO , BULLICH SÉBASTIEN , DUCROCQ FABIEN , GRALAND MARION , OLIVRY CLARA , LAYE SOPHIE , GUIARD BRUNO , FIORAMONTI XAVIER
Published: June 2020

Type-2 Diabetes mellitus (T2DM) is mainly characterized by insulin-resistance and often accompanied by comorbidities such as major depressive disorders (MDD). Indeed, patients with T2DM are twice more likely to suffer from MDD and clinical studies have shown that insulin resistance is positively correlated with the severity of depressive symptoms. In preclinical studies, we and others showed that mice fed a High Fat Diet (HFD) for 16 weeks display both hallmarks of T2DM including increased basal blood glucose level and impaired glucose tolerance and anxio-depressive-like behaviors. We also showed that the insulin-sensitizing agent metformin reverses HFD-induced impaired glucose homeostasis and emotional alterations. As the serotonergic (5-HT) system in the brain is one of the major neuronal networks involved in the pathophysiology of MDD, we hypothesized that insulin resistance at the level of 5-HT neurons contributes to the development of mood disorders in a context of T2DM. Using patch-clamp electrophysiology, we show that HFD feeding impairs the excitability of dorsal Raphe 5-HT neurons while this effect is reversed by metformin treatment. Our data also show that local application of insulin increases firing rate of 5-HT neurons in chow fed animals and this is consistent with our histochemical observations indicating that the dorsal Raphe is endowed with a high density of insulin positive cells. Additionally, preliminary data suggest that intranasal or intra-Raphe insulin injection decreases anxiety in the elevated plus maze test. Our current work aims at evaluating the intranasal and intra-Raphe insulin injection in mice lacking the insulin receptor specifically in 5-HT neurons on emotional behaviors. Altogether, this study suggests that insulin action onto dorsal Raphe 5-HT neurons is important to control emotional behaviors and that insulin-resistance in these neurons is critical in the development of T2DM-associated mood disorders.

Disclosure

H. Martin: None. S. Bullich: None. F. Ducrocq: None. M. Graland: None. C. Olivry: None. S. Laye: None. B. Guiard: None. X. Fioramonti: None.

Funding

Société Francophone du Diabète; INRAE-Région Nouvelle Aquitaine
1759-P: Prostaglandin PGE2 Receptor EP4 Signaling Deficiency in Microglia Reduces Weight Gain with Transient Glucose Improvements in Diet-Induced Obesity
Authors: NIRAULA ANZELA , FASNACHT RACHAEL , NESS KELLY M. , FREY JEREMY , DORFMAN MAURICIO D. , THALER JOSHUA
Published: June 2020

High fat diet (HFD) triggers a microglial inflammatory response in the mediobasal hypothalamus that is associated with weight gain and glucose dysregulation. Microglia depletion or selective disruption of the NFkB signaling pathway in microglia during HFD prevents the inflammatory response, and reduces food intake and weight gain. Here, we investigated the role of microglial prostaglandin PGE2 receptor 4 (EP4) signaling on the inflammatory response to HFD and metabolic outcomes. Compared to their wildtype counterparts, microglia-specific EP4 knockout (MG EP4-KO) mice on HFD showed reduced weight gain and improved glucose tolerance at 5 weeks. However, the two groups did not differ in glucose tolerance at 12 weeks, despite the MG EP4-KO mice maintaining lower body weight. This decoupling of body weight and glucose impairment indicates a progressive deterioration of glucose homeostasis associated with EP4 deficiency in microglia. Overall, these findings unravel a complex and dynamic interplay between EP4 signaling in microglia, weight gain and glucose imbalance in HFD.

Disclosure

A. Niraula: None. R. Fasnacht: None. K.M. Ness: None. J. Frey: None. M.D. Dorfman: None. J. Thaler: None.

Funding

National Institutes of Health (R01DK119754)
1767-P: Intranasal Naloxone Prevents the Development of Hypoglycemia-Associated Autonomic Failure (HAAF) in Susceptible Individuals
Authors: MANAVALAN ANJALI , MATHIAS PRIYANKA M. , ALEKSIC SANDRA , YIMAGOU ERIC LONTCHI , CAREY MICHELLE , MOY JOY , PATEL TULSI , KUO BRYAN , SANDU OANA , SHAMOON HARRY , HAWKINS MEREDITH
Published: June 2020

Repeated hypoglycemic episodes can cause HAAF, i.e., blunted counter-regulatory hormone responses. We have shown that activating opiate receptors can induce HAAF in healthy participants subjected to subsequent experimentally induced hypoglycemia. However, we have noted inter-individual variability in the development of HAAF. In our current study we evaluated the effect of blocking opioid receptors with intranasal naloxone in individuals at risk for HAAF.

To identify people susceptible to HAAF, fourteen nondiabetic subjects (13M, age 43.4±8.0, BMI 25.5±2.19 kg/m2) underwent two 120-minute hyperinsulinemic hypoglycemic (nadir 54 mg/dl) clamp studies on Day 1 and a third hypoglycemic clamp study on Day 2. Nine individuals developed HAAF, defined as ≥20% reduction in average epinephrine (EPI) levels (90-120 minutes) between the first and third episodes of hypoglycemia (858 ±161 vs. 494±108 pg/ml, p=0.008).

The HAAF-prone subjects underwent similar 3-episode hypoglycemic studies on two subsequent occasions with hourly placebo (randomized, double-blinded) or naloxone (4 mg) on Day 1. While average EPI levels declined from the first to the third hypoglycemic episode by 42% with placebo (p=0.01), levels did not differ with intranasal naloxone (p=0.67). Similarly, plasma glucagon levels decreased significantly from the first to the third hypoglycemic episode with placebo but were maintained with naloxone (Glucagon 1st vs. 3rd episode: Placebo 31.5±8.07 vs. 21.6±5.3 mg/ml, p=0.03; Naloxone 30.5±7.42 vs. 27.0±6.9 mg/ml, p=0.41). Naloxone did not alter plasma cortisol responses or hypoglycemic symptoms.

This confirms previous reports of inter-individual variability in development of HAAF. Administration of intranasal naloxone during antecedent hypoglycemia prevented HAAF in susceptible individuals. Inhaled naloxone may offer a feasible approach to ameliorate HAAF in targeted individuals at risk for hypoglycemia-related adverse events.

Disclosure

A. Manavalan: None. P.M. Mathias: None. S. Aleksic: None. E. Lontchi Yimagou: None. M. Carey: None. J. Moy: None. T. Patel: None. B. Kuo: None. O. Sandu: None. H. Shamoon: None. M. Hawkins: None.

Funding

National Institutes of Health (R01DK079974)
177-LB: Low 25-Hydroxy Vitamin D Is Associated with Arterial Stiffness in Type 2 Diabetes Mellitus
Authors: MA BINGWEI , CHEN YAO , WANG XINGCHUN , QU SHEN
Published: June 2020

Background: The aim of this study was to investigate the association between 25-hydroxyvitamin D and arterial stiffness assessed by brachial-ankle pulse wave velocity (baPWV) in type 2 diabetes mellitus(T2DM).

Methods: We identified 1335 diabetes patients from the Department of Endocrinology, Shanghai Tenth People’s Hospital from 1 November 2017 to 2 November 2019. 603 T2DM patients were included in the cross-sectional study. Participants were divided into two group: AS group (baPWV≥1700cm/s) and the control group (baPWV<1700cm/s). Linear correlation was used to evaluate the relationship between 25-hydroxyvitamin D and baPWV. Multiple linear regression and logistic regression analysis were adopted to investigate risk factors for AS.

Results: 1) Patients with AS tended to be older and had a longer duration of diabetes than control group (P<0.001 and P=0.013). Patients with AS were more likely to have diabetic retinopathy and diabetic peripheral neuropathy than the control (56% vs.43%, P=0.002; 66% vs.52%, P=0.001). 2) Compared to patients without AS, patients with AS showed lower 25-hydroxyvitamin D and higher rate of 25-hydroxyvitamin D deficiency than control group (P=0.020 and P=0.011). 3) No matter in all subjects, males or females, baPWV levels were negatively associated with 25-hydroxyvitamin D levels (r=-0.119, P=0.004; r=-0.107, P=0.044; r=-0.128, P=0.043). 4) Multiple linear regression showed that 25-hydroxyvitamin D was the negatively influencing factor of baPWV in whole sample and males (β=-2.207, P=0.014; β=-2.207, P=0.022). Logistic regression showed that age and SBP were risk factor of AS(OR:1.072, 95% CI: 1.048- 1.097, P<0.001; OR:1.028, 95% CI: 1.017-1.038, P<0.001) while VD was protective factor of AS in T2DM patients(OR:0.987, 95% CI: 0.976-0.998, P=0.024).

Conclusion: There was a negatively relationship between 25-hydroxyvitamin D and arterial stiffness assessed by baPWV. High 25-hydroxyvitamin D was found to be a protective factor for arterial stiffness.

Disclosure

B. Ma: None. Y. Chen: None. X. Wang: None. S. Qu: None.

Funding

National Key R&D Program of China (2018YFC1314100, 2016YFC1305600); Shanghai Outstanding Academic Leaders Plan (049)
177-OR: Estimating the Costs of Diabetes Complications in People <65 Years in the U.S. Using Panel Data
Authors: YANG WENYA , CINTINA INNA , HOERGER THOMAS J. , NEUWAHL SIMON , SHAO HUI , LAXY MICHAEL , ZHANG PING
Published: June 2020

Reliable, accurate, updated cost estimates of treating diabetes complications are needed to assess the financial gain of efforts to prevent and delay diabetes complications and for parameterizing diabetes cost-effectiveness simulation models. Using large longitudinal and geographically diverse claims data from Optum de-identified Normative Health Information dataset between 2007 and 2016, we estimated the annual per-person cost associated with all the main complications in people with diabetes aged <65 years. We identified persons with diabetes using diagnosis codes and distinguished type 1 (T1DM) and type 2 (T2DM) diabetes using both diagnose codes and type of medications. Complications were identified using diagnosis, procedure, or Diagnosis Related Group codes. Longitudinal panel data with a follow-up time of one to ten years were constructed. Costs in both the first and in subsequent years for a complication were estimated using individual fixed-effects models. All costs were adjusted to 2016 dollars. Our final sample included 47,166 persons with T1DM and 608,237 with T2DM. On average, compared to T2DM, the T1DM sample had a longer follow-up time (3.2 vs. 3.0 years) and was younger (39 vs. 52 years). About 46% of T1DM and 30% of T2DM samples had at least one complication. The most common complications were neuropathy, nephropathy, and history of these conditions. The estimated average costs for the three most costly conditions in the first vs. subsequent years were: end stage renal disease ($73,534 vs. $97,431 for T1DM; $94,231 vs. $98,981 for T2DM), congestive heart failure ($41,681 vs. $14,855 for T1DM; $31,202 vs. $7,062 for T2DM), and myocardial infarction ($40,899 vs. $9,496 for T1DM; $45,251 vs. $8,572 for T2DM). For both diabetes types, retinopathy and neuropathy tend to have the lowest costs. Our study provides the most updated and reliable cost estimates for evaluation of the long-term cost-effectiveness of interventions for preventing and managing diabetes.

Disclosure

W. Yang: None. I. Cintina: None. T.J. Hoerger: Research Support; Self; Centers for Disease Control and Prevention, Centers for Medicare and Medicaid Services. S. Neuwahl: None. H. Shao: None. M. Laxy: None. P. Zhang: None.

Funding

Centers for Disease Control and Prevention (200-2016-92270)