bluuebunny/crossref_metadata_2025_split huggingface dataset:
202-OR: Early De Novo DNA Methylation Patterning Regulates Adult Beta-Cell Function and Survival Programs
Authors: PARVEEN NAZIA , WANG JEAN K. , HAM ALEXANDER P. , DHAWAN SANGEETA 
Published: May 2022

DNA methylation is an important epigenetic mechanism that controls transcriptional programs to direct cell phenotype. Dynamic, stage-specific changes in DNA methylation coordinate the gene-expression patterns associated with cell differentiation throughout development. Our prior work has shown that the establishment of new DNA methylation patterns is essential for functional maturation of beta cells in postnatal life, while maintenance of existing methylation patterns sustains beta cell identity. While maintenance of DNA methylation in pancreatic progenitors is essential for their survival during differentiation, the requirement for new DNA methylation patterns in pancreatic progenitors has not been investigated. To address this, we generated pancreatic progenitor specific ablation of the de novo DNA methyltransferase, Dnmt3a, an enzyme that establishes new DNA methylation patterns, using Pdx1-Cre (Dnmt3aPKO) . RNA-sequencing of the Dnmt3aPKO islets reveals that early loss of Dnmt3a alters the beta cell identity, function, and survival programs. In addition, we observe the upregulation of genes associated with dopamine signaling and disruption of imprinted gene expression in the KO islets. Several of these gene-expression changes are distinct from those observed upon beta cell specific ablation of Dnmt3a, suggesting the unique contribution of early patterning to adult beta cell phenotype. Genome-wide profiling of DNA methylation patterns using reduced representation bisulfite sequencing (RRBS) in the Dnmt3aPKO islets points to disruption of similar programs associated with cell identity and function. These data also reveal interesting differences in the DNA methylation patterns in male and female Dnmt3aPKO mice. Collectively, our data suggest that stage specific DNA methylation patterning during beta cell differentiation controls specific aspects of the mature beta cell phenotype.

Disclosure
N.Parveen: None. J.K.Wang: None. A.P.Ham: None. S.Dhawan: None.

Funding
National Institutes of Health (R01-DK120523)
205-LB: ERK Is Required for LPS-Induced TLR4 Internalization in Macrophages
Authors: CHANG PHOEBE F. , REYNA SARA M. 
Published: May 2022

Insulin resistance is associated with circulating levels of lipopolysaccharide (LPS) released from the gut. Animal and human studies demonstrate that obesity increases gut permeability leading to elevated plasma LPS levels resulting in inflammation and metabolic dysfunction. Binding of LPS to Toll like receptor 4 (TLR4) leads to the internalization and trafficking of TLR4, inducing activation of downstream signaling pathways. TLR4 internalization in macrophages leads to activation of pro-inflammatory signaling pathways and production of factors linked to the development of insulin resistance. The extracellular signal-regulated kinase 1 and 2 (ERK1 and 2) are activated downstream of TLR4 and this activation is associated with insulin resistance. We hypothesized that ERK1 and ERK2 regulate TLR4 internalization in macrophages that are exposed to LPS. We examined whether inhibition of ERK activity blocks LPS-mediated internalization of TLR4 in bone marrow derived macrophages (BMDM) .We used siRNA to knockdown ERK1, ERK2, or both. BMDM were treated with LPS (100 ng/ml, 6hr) . Loss of cell surface TLR4 expression was measured by flow cytometry as a readout for TLR4 internalization. LPS decreased TLR4 surface expression by 31.3%, but knockdown of ERK1, ERK2, or both prevented LPS-induced decrease of TLR4 surface expression (5.6%, no decrease, 0.9%, respectively) . In addition, knockdown of either ERK1, ERK2, or both in RAW264.7 cells prevented LPS-induced activity of Rab5, the early endosomal protein associated with TLR4 translocation.
In summary, ERK regulates TLR4 endocytosis and trafficking in macrophages. We propose that ERK positively regulates LPS-mediated TLR4 internalization and inhibition of ERK signaling will protect against insulin resistance.

Disclosure
P. F. Chang: n/a. S. M. Reyna: n/a.

Funding
National Institutes of Health (SC2GM127272)
213-LB: Combined Oral Contraceptives Drive Inactivity and Hepatic Mitochondrial Oxidative Stress in Female Mice
Authors: FULLER KELLY , MCCOIN COLIN S. , STIERWALT HARRISON , THYFAULT JOHN P. 
Published: May 2022

Background: It is well established that compared to age-matched men, pre-menopausal women are protected against metabolic disease (obesity, diabetes, hepatic steatosis) . However, both epidemiological data on menopause and pre-clinical ovariectomy models show that disruption of estrogen signaling increases disease susceptibility. Combined oral hormonal contraceptives (OC) induce loss of normal estrogen cycling, and despite widespread use, the impact of OCs on metabolic health and disease remains poorly understood.
Methods: Female C57BL/6J mice (7-8 wks old) were treated with high-fat diet (HFD; CON) or HFD + OCs (2mg estradiol/kg HFD, 200mg progestin/kg HFD; OC) for 12 wks. In each treatment group, mice either remained sedentary or had access to voluntary running wheels (VWR; n=12 per group) . Liver and skeletal muscle mitochondrial function (respiratory capacity, H2O2, coupling) were measured along with body composition, glucose tolerance, energy expenditure, and intrahepatic triglyceride.
Results: OCs decreased VWR, spontaneous physical activity, and total energy expenditure. OC treated mice had lower adiposity and hepatic triglyceride compared to CON mice, with no difference in glucose tolerance. Despite similar maximal respiratory capacity, OCs induced robust increases in hepatic mitochondrial H2O2 across all respiratory states and with both fat and carbohydrate-based substrates. There were no differences in muscle mitochondrial outcomes.
Conclusions: When combined with HFD, OC use lowers physical activity and energy expenditure while inducing tissue-specific effects on mitochondrial function in mice. These data provide pause for use of OCs in the context of elevated metabolic disease risk. Future work should explore whether OC-induced changes in physical activity blunts the ability of exercise to prevent or treat insulin resistance and steatosis and whether prolonged OC exposure leads to liver injury via elevated oxidative stress.

Disclosure
K. Fuller: None. C. S. Mccoin: None. H. Stierwalt: None. J. P. Thyfault: None.

Funding
AHA (20PRE35120098) , VA Merit Review Grant (1I01BX002567-01)
217-LB: Hepatic Mig6 Ablation Restores Glucose Homeostasis during Diet-Induced Obesity and Aging
Authors: IRIMIA-DOMINGUEZ JOSE M. , BLOOM-SALDANA ELIZABETH A. , FUEGER PATRICK T. 
Published: May 2022

Hepatic epidermal growth factor receptor (EGFR) expression and activation are decreased during steatosis in humans and animal models of obesity. Restoring EGFR activation in obesity-induced endoplasmic reticulum (ER) stress and diabetes is a potential therapy to improve liver function and metabolism in type 2 diabetes (T2D) . Mitogen-inducible gene 6 (Mig6) is an inducible feedback inhibitor of EGFR activity; we previously observed that liver Mig6 expression was increased during obesity-induced insulin resistance in mice and by both pharmacological- and fatty acid-driven ER stress in hepatocytes, leading to decreased EGF-mediated EGFR activation. To test Mig6 as a potential target to treat insulin resistance in T2D, we examined liver-specific Mig6 knockout mice (LKO) and their littermate controls (CON) . LKO mice exhibited reduced liver fat content and normalized glycogen storage with diet-induced obesity. In addition, we observed enhanced hepatic insulin action and whole-body glucose tolerance, due to increased hepatic glucose uptake, as determined by hyperinsulinemic-euglycemic clamps. Using whole transcriptome sequencing, peroxisomal and mitochondrial beta-oxidation genes were decreased by Mig6 ablation, correlating with a compromised gene expression of liver gluconeogenic enzymes. Moreover, analysis of mice aged for one year, a different physiological insult causing insulin resistance and liver oxidative stress, we were able to recapitulate the main findings as with diet-induced obesity: decreased gluconeogenesis, enhanced liver glucose uptake leading to increased glucose tolerance. Moreover, in aged LKO mice, liver oxidative stress markers were reduced. In conclusion, we postulate that hepatic Mig6 deficiency partially reverts the effects of diabetes by decreasing hepatic glucose output and reducing cellular oxidative stress, thereby establishing Mig6 as a therapeutic candidate for treating insulin resistance in obesity and T2D.

Disclosure
J. M. Irimia-dominguez: None. E. A. Bloom-saldana: None. P. T. Fueger: Consultant; Protomer Technologies.
218-LB: Remission of Type 2 Diabetes after Weight Loss in "Normal" Weight People—The ReTUNE Study
Authors: TAYLOR ROY , IRVINE KEATON M. , BARNES ALISON C. , KELLY TARA L. , CLARK LUCY G. , HOLLINGSWORTH KIEREN G. , AL-MRABEH AHMAD , HOLMAN RURY R. , ROMERES DAVIDE , COBELLI CLAUDIO 
Published: May 2022

Background and aims: Mechanisms underlying weight loss induced remission of type 2 diabetes (T2DM) have been reported only in people with BMI &gt;27kg/m2. The Personal Fat Threshold hypothesis postulated that the same mechanisms in people with BMI &lt;27kg/m2. Pathophysiology during stepwise dietary weight loss was studied in this group.
Methods: People with T2DM (13/20 female, mean (±1SD) 59.3±7.1 years, BMI 24.8±1.7kg/m2) were studied before and after up to three 5% weight loss cycles, each comprising a 2-4 week low energy diet (800kcal/day formula meal replacements plus non starchy vegetables) , followed by 4-6 weeks of weight maintenance. All hypoglycemic agents were stopped before weight loss. Studies were repeated at 12 months. Outcomes were compared to normoglycemic matched controls (n=20) . Intrahepatic and intrapancreatic fat was quantified by magnetic resonance, and a standard meal test assessed insulin secretion by Disposition Index (DI) .
Results: 70% achieved remission (HbA1c &lt;6.5%; 14/20) , accompanied by decrease in adipose tissue distress (Median (IQR) PAI-1 8.64 (6.97-11.01) to 5.99 (3.94--7.73) p&lt;0.02 [control 4.87 (3.49-7.03] ng/ml; GDF-15 591 (439-872) to 444 (395-554) [446 (383-614] pg/ml p&lt;0.05. Between baseline and 12 months, decrease was (mean±SE; control data in square brackets) : BMI 24.8±0.4 to 22.5±0.4 kg/m2 (p&lt;0.0001) [21.5±0.5]; total body fat 32.1±1.5 to 27.6±1.8% (p&lt;0.0001) [24.6±1.5]; Fasting plasma insulin 47±6 to 24±6pmol/l (p&lt;0.001) [23±2]; liver fat 4.0±0.6 to 1.6±0.2% (p=0.02) [1.9±0.3]; plasma triglycerides 1.4±0.2 to 0.9±0.1mmol/l (p&lt;0.02) [0.9±0.1]; Intrapancreatic fat (6.1±0.5 to 5.0±0.6% (p=0.03) [4.1±0.3]. DI increased from median (IQR) 289 (183-373) to 774 (486-1709) dl/kg/min per pmol/l (p&lt;0.05) but remained subnormal [2751 (1524-3526].
Conclusion: Mechanistic changes underpinning remission after weight loss in non-obese people with T2DM are the same as in obese people. T2DM occurs if a person becomes too heavy for their own constitution, irrespective of BMI. Weight loss of ∼10% can bring about T2DM remission in people classified as having a ‘normal’ BMI.

Disclosure
R. Taylor: Speaker's Bureau; Janssen Global Services, LLC, Lilly Diabetes, Nestlé Health Science, Novartis AG. C. Cobelli: None. K. M. Irvine: None. A. C. Barnes: None. T. L. Kelly: None. L. G. Clark: None. K. G. Hollingsworth: None. A. Al-mrabeh: None. R. R. Holman: Advisory Panel; Anji Pharmaceuticals, Novartis AG, Novo Nordisk, Consultant; AstraZeneca, Research Support; AstraZeneca, Bayer AG, Merck Sharp & Dohme Corp. D. Romeres: None.

Funding
Diabetes UK (17/0005645)
220-OR: Characteristics Associated with Adding Insulin in Type 2 Diabetes in the Glycemia Reduction Approaches in Diabetes (GRADE) Study
Authors: MARTIN CATHERINE , HOLLANDER PRISCILLA A. , KRAUSE-STEINRAUF HEIDI , KAZEMI ERIN , BUTERA NICOLE M. , AHMANN ANDREW , FALCK-YTTER CORINNA , FATTALEH BASMA , JOHNSON MARY L. , KILLEAN TINA , LAGARI VIOLET , LARKIN MARY E. , LEGOWSKI ELIZABETH , RASOULI NEDA , WILLIS HOLLY ,   GRADE RESEARCH GROUP
Published: May 2022

Delaying insulin when indicated in type 2 diabetes (T2D) prolongs exposure to hyperglycemia. In the GRADE study, adults (N=5047) with T2D (duration &lt;years, metformin monotherapy, HbA1c 6.8 to 8.5%) were randomly assigned to glimepiride, sitagliptin, liraglutide or insulin glargine U-100 and followed for mean and median of 5 years. The protocol specified addition of basal insulin glargine (non-insulin groups) or prandial insulin aspart (glargine group) ≤6 weeks after confirming the outcome of A1c&gt;7.5%. Written consent included acceptance of injectable medications, including insulin, and to add or intensify insulin therapy after outcome confirmation. Among 2387 participants with confirmed A1c&gt;7.5%, 834 (35.0%) added insulin within 6 weeks, 698 (29.1%) after 6 weeks, and 855 (35.8%) never. When we did a comparison by time of initiation, there were differences by age, racial group, outcome A1c and assigned treatment group. Participants never adding insulin were more likely to be older, Black/African American, or assigned to glargine. Those who added insulin were more likely to have higher confirmation A1cs or be assigned to sitagliptin. Analyses to explore investigator and participant factors influencing decisions to delay or not add insulin despite suboptimal glucose control are underway.

Disclosure
C. Martin: Advisory Panel; Nevro Corp. P.A. Hollander: None. H. Krause-Steinrauf: None. N.M. Butera: None. A. Ahmann: Advisory Panel; Medtronic. C. Falck-Ytter: None. B. Fattaleh: None. M.L. Johnson: Research Support; Abbott Diabetes, Dexcom, Inc., Insulet Corporation, Jaeb Center for Health Research, Lilly, Medtronic, Novo Nordisk, Sanofi. V. Lagari: None. M.E. Larkin: None. N. Rasouli: Advisory Panel; Eli Lilly and Company, Novo Nordisk, Sanofi. Research Support; Allergan, Eli Lilly and Company, Novo Nordisk. H. Willis: Research Support; Abbott Diabetes, Sanofi.

Funding
National Institute of Diabetes and Digestive and Kidney Diseases (U01DK098246, U34-DK-088043)
224-OR: Antisense Oligonucleotide–Mediated Restriction of Mitochondrial Pyruvate Metabolism Limits DNL in High-Fat Fed C57BL6/J Mice
Authors: STROBER JORDAN W. , PAOLELLA LAUREN M. , SUH REBECCA , HUBBARD BRANDON T. , MURRAY SUE , VATNER DANIEL F. 
Published: May 2022

Increased de novo lipogenesis (DNL) contributes to lipid disturbances prevalent in insulin resistant (IR) subjects, including dyslipidemia and NAFLD; however, the specific carbon sources that maintain DNL during hepatic IR remain unknown. To address this question, antisense oligonucleotides (ASOs) targeting glucokinase (GCK) , ketohexokinase (KHK) , lactate dehydrogenase (LDHA) , and glutamic-pyruvic transaminase 2 (GPT2) were applied to restrict substrate influx from specific lipogenic precursors. Subsequently, the rates of DNL were assessed via the deuterated water method, and triglyceride (TG) content was measured in both insulin-sensitive regular chow (RC) fed and IR six-week high-fat diet (HFD) fed C57BL6/J mice. In RC mice, there were no significant differences in hepatic DNL as a result of treatment with any ASO. Furthermore, TG content remained unchanged by most ASOs in the RC cohort, but was increased in the liver with the LDHA ASO (Con: 7.4±0.2 μg/mg; LDHA: 16.9±1.2 μg/mg; p&lt;.001) and decreased in circulation with the GCK ASO (Con:84.8±6.3 mg/dL; GCK:54.5±5.8 mg/dL; p&lt;.01) . In the HFD cohort, DNL, hepatic TG content, and circulating TGs were all reduced by restricting lipogenic carbon entry from glucose (GCK ASO vs. Con: DNL -69.2%±3.6, p&lt;.01; Hep TG -59.2%±4.4, p&lt;.05; Cir TG -35.7%±3.7, p&lt;.001) , lactate (LDHA ASO vs. Con: DNL -33.1%±9.1, p&lt;.05; Hep TG -25.8%±7.4, p&lt;.05; Cir TG -18.0%±8.0, NS) , and alanine (GPT2 ASO vs. Con: DNL -48.1%±9.8, p&lt;.01; Hep TG -45.5%±8.9, p&lt;.01; Cir TG -23.2%±5.3, p&lt;.01) .
Conclusion: RC fed mice maintain DNL despite restricting specific carbon precursors; however, in HFD-fed mice, glucose, lactate, and alanine are all necessary substrates for maintaining aberrantly elevated rates of DNL, and any substrate restraint is associated with reduced DNL. These findings expound how specific carbon fluxes support DNL in IR and may inform the development of novel therapies for dyslipidemia.

Disclosure
J.W.Strober: None. L.M.Paolella: None. R.Suh: None. B.T.Hubbard: None. S.Murray: Employee; Ionis Pharmaceuticals. D.F.Vatner: None.

Funding
NIH T32 (DK007058) NIH R (DK124272)
226-LB: Genetic Variation of Human Myokine Signaling Is Dominated by Biologic Sex and Sex Hormones
Authors: VAN CASSANDRA , SELDIN MARCUS 
Published: May 2022

Skeletal muscle physiology is a critical determinant and therapeutic target for type 2 diabetes. Proteins secreted from skeletal muscle, termed myokines, allow the muscle to communicate with other key metabolic organs and several have been established as causal drivers of processes such as exercise and lipid metabolism. Yet, the genetic architecture, regulation, and functions of myokines, as well as degree of conservation of these communication circuits remains inadequately understood. In this study, we performed the first targeted genetic survey of myokine gene regulation and cross-tissue signaling in 310 humans where sex as a biological variable was emphasized. While expression levels of a majority of myokines and cell proportions within skeletal muscle showed little differences between males and females, nearly all significant cross-tissue enrichments operated in a sex-specific or hormone-dependent fashion. Estrogen signaling specifically in muscle showed striking enrichments with myokines, independent of sex-specific signaling. To define the causal roles of estrogen signaling on myokine gene expression and functions, we generated male and female mice lacking estrogen receptor α (Esr1) specifically in skeletal muscle and integrated global RNA-Sequencing with human data. These analyses highlighted mechanisms of sex-dependent myokine signaling conserved between species, such as myostatin enriched for divergent substrate utilization pathways between sexes, and GPX3 as a male-specific link between glycolytic fiber abundance and hepatic inflammation. Collectively, we provide the first genetic survey of human myokines and highlight sex and estrogen receptor signaling as critical variables when assaying myokine functions and how changes in cell composition impact other metabolic organs. This study is currently available on bioRxiv: https://www.biorxiv.org/content/10.1101/2022.01.20.477045v1

Disclosure
C. Van: None. M. Seldin: None.
229-LB: G6PC2, a GWAS Locus for Fasting Glucose and HbA1c, Regulates Glucose-Suppression of Glucagon Secretion in a-Cells
Authors: BAHL VARUN , MAY CATHERINE L. , PANCREAS ANALYSIS PROGRAM HUMAN , KAESTNER KLAUS H. 
Published: May 2022

The G6PC2 locus, encoding the catalytic subunit of the islet-specific glucose-6-phosphatase enzyme, is an established risk locus for HbA1c and fasting glucose levels. A critical role of glycolytic flux in regulating glucagon secretion was established through manipulation of the activity and expression of the glycolytic enzyme glucokinase (GCK) which controls the setpoint for glucose-suppression of glucagon secretion (GSGS) . G6PC2 opposes the action of GCK by hydrolyzing glucose-6-phosphate back to glucose and creating a futile substrate cycle. Previous studies from germline and β-cell-specific G6PC2 null mice have demonstrated that G6PC2 ablation reduces glucose-6-phosphatase activity, glucose cycling, and glycolytic flux, resulting in a leftward shift of dose-response curve for glucose-stimulated insulin secretion. Through cell type specific chromatin accessibility maps of sorted human islet cells, we established that G6PC2 risk SNPs are in open chromatin regions in human α-cells, suggesting that they might not only affect β-cell but also α-cell expression of this critical gene. To test whether α-cell G6PC2 impacts glycemic control, we derived a new mouse line that induces ablation of the first three exons of G6PC2 after Cre activation – resulting in a null allele (G6pc2 loxP/loxP; Gcg-CreERT2; referred to as “α-G6PC2KO”) . Assessment of glucagonemia during glucose tolerance tests revealed a decrease in glucagon levels in α-G6PC2KO mice relative to controls. Intriguingly, fasting blood glucose levels were significantly lower in α-G6PC2KO mice. α-G6PC2KO mice recovered more slowly from hypoglycemia during an insulin tolerance test, and presented lower glucagon levels relative to control mice following the insulin bolus. α-G6PC2KO islets secreted less glucagon relative to control islets when incubated in low-glucose media, independent of alterations in insulin secretion or islet hormone content. Collectively, our data demonstrate that the G6PC2 locus impacts glycemic control via its action in α-cells.

Disclosure
V. Bahl: None. C. L. May: None. Human pancreas analysis program: n/a. K. H. Kaestner: None.
23-LB: A Study of Taste among Type 2 Diabetes Mellitus Patients with Autonomic Neuropathy at a Tertiary Care Centre in North India
Authors: KUSHWAHA JITENDRA , SINGH ARCHANA , GIRI RICHA , GUPTA VISHAL K. 
Published: May 2022

Introduction: Type 2 diabetes mellitus (T2DM) is a multifactorial, complex disease associated with chronic hyperglycemia, resulting from the interplay of genetic, environmental, and epigenetic factors (1) . Diabetic autonomic neuropathy (DAN) is one of the common complication in diabetes. The taste threshold affected by various factors such as age, ethnic backgrounds, drugs, local and systemic diseases, consumption of alcohol, smoking, and tobacco chewing . The present study is undertaken with the objectives to compare the alteration in taste threshold for four primary sensations in Type 2 DM with autonomic neuropathy.
Material and methods: 60 patients of T2DM with autonomic neuropathy and 60 healthy control were taken for the study. Autonomic neuropathy were assessed clinically. Chemical taste test using four solutions of basic tastes (sweet, sour, salty, bitter) were done. 5 ml sample of each solution was provided whose nature was kept unknown to the subject and asked to hold it there for 5 seconds before spitting the solution into the cup.
Result: Out of 60 patients 26 (43.3%) were having taste dysfunction for sweet compared to 12 (20%) out of 60 healthy controls with p value 0.006. Taste dysfunction for sweet was significant in T2DM with uncontrolled hyperglycemia i.e mean HbA1c 10.83+/- 3.17 whereas in T2DM patients whose taste were preserved had mean HbA1c 8.05+/- 1.87 . The taste dysfunction in T2DM patients was not related to gender, disease duration, and type of treatment taken.
Conclusion: The study found a significant correlation between taste dysfunction, HbA1C level and blood sugar fasting level in T2DM patients. The taste dysfunction was mainly for sweet. Sour and bitter did not show any difference in case groups compared to controls.

Disclosure
J. Kushwaha: None. A. Singh: None. R. Giri: None. V. K. Gupta: None.