bluuebunny/crossref_metadata_2025_split huggingface dataset:
3-OR: Hepatocyte Gdf15 Is Sufficient for Increased Plasma GDF15 Levels and Body Weight Constraint during High-Fat–Diet Feeding and Restores Insulin Sensitivity in a Liver-Specific Manner
Authors: XIE BINGXIAN , MURALI ANJANA , VANDEVENDER AMBER , GIL SILVA AGUSTIN A. , BELLO FIONA , SIPULA IAN J. , DEDOUSIS NIKOLAOS , ALDER JONATHAN , JURCZAK MICHAEL J. 
Published: May 2022

Growth and differentiation factor 15 (GDF15) is a stress-induced secreted protein whose plasma levels are increased in obese patients. Recombinant GDF15 reduces body weight (BW) and improves glycemia in obese models, which is largely attributed to central action of GDF15 to suppress feeding. Despite this, the tissue specific sites of GDF15 production during obesity are unknown and the potential for local action is understudied. To determine the hepatocyte specific contribution of Gdf15 to changes in circulating GDF15 and effects on obesity, we utilized a Gdf15 KO-first mouse strain where endogenous expression of Gdf15 can be restored in a tissue specific fashion via Flp recombinase (FLP) . Gdf15 KO-first mice received AAV8-TBG-Gfp (KO+GFP) or AAV8-TBG-Flp (liver specific Gdf15 expression; KO+FLP) and were fed HFD for 12 weeks, alongside age-matched C57BL6 mice (WT) . Plasma GDF15 levels were undetectable in KO+GFP and similar between KO+FLP and WT HFD. BW was reduced in KO+FLP compared with KO+GFP and was not different from HFD WT. Interestingly, differences in BW were due to greater changes in lean (LM) compared with fat mass (FM) . Feeding was reduced in KO+FLP when expressed per mouse but not when expressed per g BW or LM, nor when the relationship between BW/LM and feeding were assessed by ANCOVA. Plasma insulin levels were increased in KO+GFP compared with WT HFD and markedly lower in KO+FLP compared with KO+GFP and WT HFD. Hyperinsulinemic euglycemic clamps demonstrated improved insulin sensitivity in KO+FLP due solely to changes in liver insulin action. These data suggest that feeding alone did not account for the changes in BW or insulin resistance, which would favor loss of FM and a generalized improvement in whole-body insulin sensitivity. They also demonstrate that hepatocyte Gdf15 is sufficient for obesity-associated increases in circulating GDF15 and suggest the potential for local effects of GDF15.

Disclosure
B.Xie: None. A.Murali: None. A.Vandevender: None. A.A.Gil silva: None. F.Bello: None. I.J.Sipula: None. N.Dedousis: None. J.Alder: None. M.J.Jurczak: None.

Funding
Pittsburgh Foundation (MR2020 109502)
300-OR: A Modular Massively Parallel Reporter Assay Uncovers Context-Specific Activity of Diabetes-Associated Regulatory Elements
Authors: TOVAR ADELAIDE 
Published: May 2022

Recent genome-wide association studies (GWAS) have identified hundreds of signals associated with type 2 diabetes (T2D) , most (∼90%) of which are found in noncoding regions of the genome enriched in tissue-specific regulatory regions. However, most GWAS signals span hundreds of tightly linked variants, and predicting each of their functional effects is difficult. Consequently, progress to translate associations to mechanisms has proceeded slowly. Here, we have designed a massively parallel reporter assay (MPRA) to systematically screen enhancer activity across a panel of genomic fragments, including (1) islet-specific TSSs defined by cap analysis of gene expression (CAGE) and (2) allelic pairs for over 10k T2D- and metabolic trait-associated variants. We cloned this library up- or downstream of a reporter gene driven by a synthetic housekeeping promoter (SCP1) or the human insulin (INS) promoter, together enabling enhancer activity measurements across different promoter and enhancer contexts. We examined expression bias within our library (FDR &lt; 0.05) and found that 43% of CAGE-defined oligos were marked by positional bias (n = 565/1,305) , skewed towards higher expression in the upstream position (n = 539/565) . In contrast, two unique subsets of GWAS oligos displayed either positional (n = 702/11,656) or promoter (n = 698/11,656) bias. Oligos with positional bias were evenly split between the two cloning positions, while most promoter-biased oligos were more highly expressed when paired with the INS promoter (n = 512/698) . To identify sequence features associated with promoter bias, we performed Lasso regression using a set of 562 genomic annotations. This analysis revealed that presence of HNF1 motifs was associated with higher enhancer activity in the context of the INS promoter. Together, these results indicate that reporter construct design clearly influences assayed regulatory activity, particularly with use of tissue-relevant promoters.

Disclosure
A.Tovar: None.

Funding
National Institutes of Health (R01DK117960, T32DK101357)
301-OR: Modeling the Type 2 Diabetes–Associated ABCC8 Arg1420 His Mutation using iPSC–Derived Pancreatic Islet-Like Cells
Authors: NAIR ANUP K. , TRAURIG MICHAEL , SUTHERLAND JEFF , NELSON ROBERT G. , BOGARDUS CLIFTON , BAIER LESLIE 
Published: May 2022

Genetic studies in American Indians from Arizona previously identified a population specific Arg1420 His mutation in ABCC8 which causes hyperinsulinemic hypoglycemia in infancy. The His allele is carried by 3% of this population and associates with increased birthweight, suggesting increased insulin secretion in-utero, and doubles the risk of type 2 diabetes, a disease characterized by reduced insulin secretion. To model the contrary effects of this mutation, we used gene-edited induced pluripotent stem cell (iPSC) derived pancreatic islet-like cells. An iPS cell line created from an American Indian heterozygous for the mutation was gene-edited using CRISPR-CAS9 to generate iPSCs homozygous for the risk (His) and the non-risk (Arg) allele. Multiple iPSC lines for each genotype were differentiated towards pancreatic islet-like cells using a seven-stage differentiation protocol and the effect of the mutation on islet function were assessed. Higher insulin secretion was observed from the His-allele carrying islet-like cells when stimulated with low glucose (2mM) after 1 and 2 weeks of maturation, consistent with a clinical phenotype of hyperinsulinemia in His-allele carriers. Stimulation with 20mM glucose resulted in increased insulin secretion after 2 weeks of maturation only in the Arg-allele (non-risk) carrying islets while no increase was seen in the His-allele carrying islets, consistent with its role in T2D risk. Treatment with diazoxide significantly reduced insulin secretion with no difference between the genotypes. Single cell RNA sequencing of islet-like cells cultured for an extended time (4 weeks) identified many differentially expressed genes in cells with the mutant vs. reference allele that could potentially reveal the mechanism of the increased future T2D risk including G6PC2, a known negative regulator of glucose-stimulated insulin secretion, in beta-like cells and CRH, which has been linked to increased T2D risk, in alpha-like cells.

Disclosure
A.K.Nair: None. M.Traurig: None. J.Sutherland: Stock/Shareholder; Bristol-Myers Squibb Company, Johnson & Johnson, Pfizer Inc. R.G.Nelson: None. C.Bogardus: None. L.Baier: None.

Funding
NIH Intramural
304-OR: Proteomic Profile and Inflammatory Response of Islet Stressomes Released during Isolation
Authors: SARAVANAN PRATHAB BALAJI , KALIVARATHAN JAGAN , LEVY MARLON F. , KANAK MAZHAR A. 
Published: May 2022

Purpose: Islets undergo enormous stress during isolation and peri-transplant periods which causes a significant release of stressomes (exosomes released during stress) . Uncovering the biological role of such vesicles is vital for long-term graft function and survival. Here, we sought to identify the global protein content and significance of stressomes isolated during isolation in autologous islet transplantation (AIT) .
Methods: Post-transplant stress was mimicked in vitro by exposing human islets to hypoxia and cytokines (Cyt+Hyp) . Stressome protein contents were characterized by mass spectrometry and immunoblotting. Similar stressomes were also isolated from the islet infusion bag samples of AIT recipients (N=20) . The stressome protein levels were correlated with 1-year AIT outcomes. Finally, these islet stressomes were treated to another set of purified islets and macrophages to find their uptake and immune response.
Results: The mass spectrometry analysis has revealed the islet stressomes contain DAMP proteins like histones (H2B, H3, and H4) . The canonical ER stress signal plays a crucial role in the biogenesis of islet stressomes induced by (Cyt+Hyp) . Exosomes isolated from the infusion bags showed an elevated histone level in the insulin-dependent (ID) group. Moreover, high levels of DAMP proteins were positively correlated with increased post-transplant recipients insulin requirements (p=0.029) . The fluorescent confocal microscopy analysis has revealed these stressomes were easily uptaken by the macrophages and also conferred an inflammatory phenotype. Instead, the stressome treated islets showed elevated expression of innate immune receptors like TLR2 and AGTR1.
Conclusion: The study has identified the islet stressome global protein content and briefs its prospective to induce islet inflammation and graft loss in AIT.

Disclosure
P.Saravanan: None. J.Kalivarathan: None. M.F.Levy: Research Support; Vertex Pharmaceuticals Incorporated, Speaker's Bureau; AbbVie Inc., Sanofi Genzyme. M.A.Kanak: None.
307-OR: Impaired Differentiation of Trpv1-Positive Adipose Progenitors Activates Compensatory Thermogenic Mechanisms
Authors: CAMARA HENRIQUE , SHAMSI FARNAZ , LYNES MATTHEW , TSENG YU-HUA 
Published: May 2022

Brown and beige thermogenic adipocytes are specialized in energy consumption and linked to metabolic health. Canonically, thermogenic adipocytes are thought to be derived from mesenchymal PdgfRα+ adipocyte progenitor cells (APC) . We recently identified a novel population of cold-recruited APCs from the vascular smooth muscle lineage marked by the expression of the ion channel Trpv1 in mouse adipose tissue. To determine the function of the Trpv1+ APC-derived adipocytes, we impaired their adipogenic capacity by deletion of Pparγ in Trpv1-expressing cells in mice (Trpv1-Cre::Pparγ-flox) . Inhibition of de novo adipogenesis from Trpv1+ APCs resulted in modest changes in brown adipose tissue, but it caused a marked reduction in the expression of cold-induced browning genes, including UCP1, in white adipose tissue (WAT) , indicating impaired beige adipogenesis. Interestingly, Trpv1-Cre::Pparγ-flox mice did not show altered weight gain, glucose tolerance, or insulin sensitivity on either regular chow or high-fat diets. This discrepancy led us to search for potential compensatory mechanisms. We observed increased PdgfRα expression in WAT of Trpv1-Cre::Pparγ-flox animals, suggesting that impaired adipogenic capacity of the Trpv1+ APCs may trigger recruitment of the PdgfRα+ APCs. Additionally, while UCP1 expression was noticeably reduced in the beige adipocytes within WAT, the expression of transcripts involved in the UCP1-independent thermogenic pathways, including Ckmt1, Gatm, and Slc6a8, key components of the creatine futile cycle, was increased in the WAT of the Trpv1-Cre::Pparγ-flox mice upon cold exposure. Together, these data highlight a potential distinction between the thermogenic pathways mediated by adipocytes derived from the Trpv1+ versus those derived from the PdgfRα+ APCs. This functional difference of thermogenic adipocytes could be critical in designing strategies to target these cells as a therapeutic approach for metabolic diseases.

Disclosure
H.Camara: None. F.Shamsi: None. M.Lynes: None. Y.Tseng: Consultant; Cellarity.

Funding
National Institutes of Health (R01DK102898)
310-OR: Histone Variant H2A.Z-Mediated Chromatin Organization Regulates Brown Adipocyte Thermogenic Function
Authors: ZHANG YANG , ZHENG RONGBIN , WANG CHIH-HAO , DARCY JUSTIN , CHEN KAIFU , TSENG YU-HUA 
Published: May 2022

Obesity accounts for a substantial and growing burden of global disease, leading to high morbidity and mortality in type 2 diabetes, cardiovascular disease, and certain cancers. Brown adipose tissue (BAT) dissipates energy as heat and has been proposed as a therapeutic target to combat obesity. The unique thermogenic capacity of BAT is primarily conferred by uncoupling protein 1 (Ucp1) , which dissipates energy by uncoupling the proton motive force from ATP generation. To identify novel transcriptional regulators of Ucp1, we performed an unbiased CRISPR/Cas9-based chromatin immunoprecipitation assay (enChIP) and identified H2A.Z as a novel trans-acting factor recruited to the promoter and enhancer of Ucp1 by β3-adrenergic receptor (β3-AR) stimulation. H2A.Z is an essential and evolutionary conserved histone variant of the canonical histone H2A. Knockdown of H2A.Z in brown adipocytes led to lower β3-AR-induced Ucp1 level and impeded the expression of other known thermogenic genes (e.g., Cidea, Pgc1a) , leading to impaired cellular thermogenesis. Mechanistically, by profiling the genome-wide distribution of H2A.Z via ChIP-seq, we observed H2A.Z was enriched at promoter and enhancer of Ucp1 upon β3-AR stimulation, which is consistent with the enChIP result. Intriguingly, we found that the average occupancy of H2A.Z in the promoter and enhancer regions of other β3-AR-upregulated genes (e.g., Zfp516, Vdr, Pck1, Gys2) was also higher than that in non-stimulated condition, indicating a global regulatory role of H2A.Z in the thermogenic program in response to β3-AR stimulation. Additionally, in the enhancer regions of β3-AR-induced genes, we also detected an increased level of H2A.Z acetylation, which is a mark of active chromatin and confers an open chromatin conformation. Taken together, these findings reveal the essential role of H2A.Z-mediated chromatin organization in BAT thermogenic function.

Disclosure
Y.Zhang: None. R.Zheng: None. C.Wang: None. J.Darcy: None. K.Chen: None. Y.Tseng: Consultant; Cellarity.

Funding
National Institutes of Health (R01DK102898)
313-OR: A Cytoskeleton-Based Mechanism Regulates Lipid Metabolism and Energy Homeostasis in Brown Adipose Tissue
Authors: AGUILLARD ASHLEY , ARRICASTRES JUNE , TZENG JOYCE , LORENZO DAMARIS 
Published: May 2022

Genetic variants in ANK2, encoding ankyrin-B (AnkB) , increase risk of obesity and type 2 diabetes in humans. We previously concluded that AnkB deficiency in white (WAT) and brown (BAT) adipose tissue (AT) of mice (AT-AnkB KO) led to lipotoxicity, WAT hypertrophy, and insulin resistance associated with age and consumption of a high-fat diet. These metabolic deficits were caused by impairments in glucose transporter GLUT4 endocytosis in WAT due to AnkB deficiency. Additionally, we found that AT-AnkB KO mice had decreased energy expenditure and lipid accumulation in BAT prior to the onset of obesity suggesting a potential role of AnkB in BAT. However, the role of AnkB in BAT and its contribution to metabolic homeostasis through BAT has not been elucidated.
We investigated the role of AnkB in mice conditionally lacking AnkB in BAT (BAT-AnkB KO) . Based on histological and transmission electron microscopy analysis, we found that young (4-month-old) BAT-AnkB KO mice have enlarged lipid droplets, lipid accumulation, and decreased mitochondrial content in BAT with no changes in BAT size, body weight, or body composition. Consistent with these findings, primary brown adipocytes from BAT-AnkB KO mice exhibited lipid accumulation suggesting that AnkB may regulate lipid homeostasis in BAT in a cell-autonomous manner. Similar to our previous findings, young BAT-AnkB KO mice also had decreases in oxygen consumption and energy expenditure. These mice also exhibit minimal changes in glucose handling and insulin tolerance at 4-months of age. Proteomic-MS analysis was performed on AnkB associated proteins from BAT which revealed that GLUT4 as well as a number of critical regulators of lipid metabolism form complexes with AnkB in BAT. In combination with this data, we will present our latest findings in gene expression of genes regulating lipid metabolism and mitochondrial function as well as cellular assays in brown adipocytes that may highlight metabolic pathways regulated by AnkB in BAT.

Disclosure
A.Aguillard: None. J.Arricastres: None. J.Tzeng: None. D.Lorenzo: None.

Funding
American Diabetes Association (1-19-JDF-081)
317-OR: The Novel Glucokinase GCK (Q26L) Gene Mutation Site Causes Diabetes
Authors: JI SHUHUI , SHU HUA , FENG WENLI , ZHEN JINYANG , QIAO JINGTING , HUANG YADI , CHEN SHANSHAN , LIU MING 
Published: May 2022

Glucokinase (GCK) mainly exists in pancreatic β-cell, hepatocytes and brain, and is the first-rate limiting enzyme in glucose metabolism pathway. The heterozygous inactivation mutation of GCK leads to Maturity-onset diabetes of the young (MODY2) , an autosomal dominant mild fasting hyperglycemia. In the monogenic diabetes registration project initiated by our research group, we found a novel inactivation mutation site of GCK gene, c.77A&gt;T (p.Q26L) . We sought to explore the pathogenic mechanism with a newly constructed mouse model.The proband was first diagnosed with diabetes at 11. Her father was diagnosed with diabetes at 32, and his mother was healthy. Genetic testing showed there was a novel heterozygous missense mutation site c.77A &gt;T in exon 1 of the GCK of the proband, which resulted in the change of the 26th amino acid from glutamine to leucine. The pedigree indicated the mutation of proband was inherited from her father, not her mother. A global knock-in GCK (Q26L) mice (GCKKI/-) model was successfully established. From 3-16 weeks, substantial group differences in fasting blood glucose, other than body weight, was observed in GCKKI/-. At 3-week, impaired glucose tolerance and lower serum insulin level of GCKKI/- mice were detected. There was no difference in insulin sensitivity and content of proinsulin and insulin in the islet of GCKKI/-. In vitro experiment, decreased glucose-stimulated insulin secretion was observed in the GCKKI/-, but no significant changes in KCl stimulated insulin secretion. Furthermore, the results of different glucose concentration stimulated insulin secretion and islet perfusion showed the insulin secretion threshold increased and the insulin secretion ability decreased in the GCKKI/-. In the GCKKI/- liver, there were no changes in the morphology, but glycogen content was reduced. The level of plasma HDL significantly increased, plasma TC and LDL levels decreased, and TG levels did not change.

Disclosure
S.Ji: None. H.Shu: None. W.Feng: None. J.Zhen: None. J.Qiao: None. Y.Huang: None. S.Chen: None. M.Liu: None.

Funding
National Natural ScienceFoundation of China (81830025,81620108004,81800733,and 81900720) , the Ministry of Science and Technologyof China (2019YFA0802502) , the Tianjin Municipal Science andTechnology Bureau (17ZXMFSY00150)
319-OR: Na+/K+ ATPase Activation Is a Conserved Mechanism for Gi/o Protein-Coupled Receptor Control of Pancreatic ß-Cell Electrical Excitability and Insulin Secretion
Authors: DICKERSON MATTHEW , DADI PRASANNA , ZABORSKA KAROLINA E. , NAKHE ARYA Y. , SCHAUB CHARLES , DOBSON JORDYN , WRIGHT NICOLE M. , LYNCH JOSHUA , SCOTT CLAIRE , JACOBSON DAVID 
Published: May 2022

Gi/o protein-coupled receptor (Gi/o-GPCR) inhibition of pancreatic β-cell Ca2+ entry limits islet insulin secretion; however, the mechanism has not been identified. As Gi/o-GPCRs activate G protein-gated inwardly rectifying K+ (GIRK) channels, we examined their contribution to β-cell Gi/o signaling. However, Gi/o-GPCR-induced β-cell GIRK currents were not observed; this was demonstrated with a pharmacological approach and by genetic knockout of the predominant β-cell GIRK channel subunit. Thus, we increased the chemical driving force for K+ movement through GIRK channels by removing extracellular K+. Interestingly, under these conditions Gi/o-GPCR-induced β-cell membrane potential (Vm) hyperpolarization was lost even though K+ channel activation could still hyperpolarize Vm, which indicated that Gi/o-GPCRs do not hyperpolarize β-cell Vm by activating K+ channels. As extracellular K+ is required for Na+/K+ ATPase (NKA) activity, we next tested and found that mouse and human β-cell NKAs were activated by Gi/o-GPCRs. Stimulation of Gi/o-GPCRs initiated cyclical β-cell NKA activity, resulting in islet Ca2+ oscillations. Moreover, paracrine activation of β-cell NKAs by intraislet δ-cell somatostatin (SST) secretion decelerated islet Ca2+ oscillations and diminished insulin secretion, which was mediated by direct activation of β-cell Gi/o-GPCRs. Gi/o signaling induced islet Ca2+ oscillations through Src tyrosine kinase-dependent NKA phosphorylation and activation; an effect that was recapitulated by stimulating insulin receptor tyrosine kinases. Whereas, islet NKA function was completely inhibited by cAMP-dependent PKA activation. Therefore, these findings reveal that NKA-mediated hyperpolarization of β-cell Vm is the primary and conserved mechanism for Gi/o-GPCR control of electrical excitability, Ca2+ handling, and insulin secretion.

Disclosure
M.Dickerson: None. D.Jacobson: None. P.Dadi: None. K.E.Zaborska: None. A.Y.Nakhe: None. C.Schaub: None. J.Dobson: None. N.M.Wright: None. J.Lynch: None. C.Scott: None.

Funding
American Diabetes Association (1-17-IBS-024) ; Vanderbilt Integrated Training in Engineering and Diabetes Grant (T32DK101003) , National Institutes of Health Grants (DK-097392 and DK-115620) , Juvenile Diabetes Research Foundation Grant (2-SRA-2019-701-S-B) , and a Pilot and Feasibility grant through the Vanderbilt Diabetes Research and Training Center Grant (P60-DK-20593) .