bluuebunny/crossref_metadata_2025_split huggingface dataset:
1878-P: Glucagon Is neither Necessary nor Sufficient for Ketosis in Mice
Authors: CAPOZZI MEGAN , COCH REILLY , WAIT JACOB B. , KOECH JEPCHUMBA J. , CAMPBELL JONATHAN , D'ALESSIO DAVID A. 
Published: June 2019

The current consensus mechanism for hepatic ketogenesis posits a key role for islet hormones, with insulin inhibiting and glucagon stimulating formation of ketone bodies. In this model the action of glucagon in the presence of low portal insulin concentrations causes ketosis. Sodium-glucose co-transporter 2 inhibitors (SGLT2i) are effective for lowering glucose in diabetic patients, and cause a relative insulinopenia. These agents have been described to increase ketone production, and have been associated with cases of diabetic ketoacidosis. SGLT2i have been recently reported to stimulate islet α-cells, suggesting the hypothesis that SGLT2i-induced glucagon secretion drives ketogenesis. To test this hypothesis, mice were given the SGLT2i dapagliflozin (dapa) in the presence or absence of glucagon signaling. Fasting-induced ketosis was used as a positive control. In both dapa-treated mice and 16 hour fasted mice, blood glucose decreased to the 4-5 mM range, β-OHB increased 2-3 fold, and NEFA rose 30-50%. Fasting had minimal effects on plasma glucagon, but dapa increased glucagon concentrations, albeit at a delayed rate compared to changes in glycemia, β-OHB, or NEFAs. Interruption of glucagon signaling with either: 1) a glucagon receptor antagonist (GRA); or 2) mice with a deletion of the proGlucagon gene had no effect on the development of ketosis in response to dapa or fasting, indicating that glucagon is not necessary for ketosis in these conditions. To test its sufficiency, glucagon (20µg/kg) was given to fed mice, 16 hour fasted mice, dapa-treated mice, and mice with insulin receptor blockade (IRB). Fasting, dapa and IRB all increased β-OHB significantly but glucagon did not add to this. In contrast, epinephrine significantly elevated ketone levels under these conditions. These findings indicate that the current model of the regulation of ketogenesis needs to be reconsidered, and that in mice glucagon does not have a significant role in this process.

Disclosure
M. Capozzi: None. R. Coch: None. J.B. Wait: None. J.J. Koech: None. J. Campbell: Research Support; Self; Eli Lilly and Company. Speaker's Bureau; Self; Merck Sharp & Dohme Corp. D.A. D'Alessio: Advisory Panel; Self; Eli Lilly and Company. Research Support; Self; Eli Lilly and Company, Merck & Co., Inc.

Funding
National Institutes of Health (F32-DK116542)
1883-P: Phosphoproteomics Reveals Mechanistic Insight into Glucagon-Mediated Inhibition of Hepatic Lipogenesis and How MEDI0382, an Oxyntomodulin-Like Peptide with Targeted GLP-1/Glucagon Receptor Activity, Reverses Steatosis
Authors: LAKER RHIANNA C. , BOLAND MICHELLE , NAWROCKI ARKADIUSZ , MATHER KARLY M. , JERMUTUS LUTZ , LARSEN MARTIN R. , TREVASKIS JAMES , RHODES CHRISTOPHER J. 
Published: June 2019

MEDI0382 is an oxyntomodulin-like peptide with targeted GLP-1/glucagon receptor activity that is in clinical development for type 2 diabetes (T2DM), NASH, and obesity and provides glycemic control and weight loss as well as marked improvements of liver outcomes in humans and mice. In mice, reductions in hepatic lipid were independent of weight loss and resulted from improved mitochondrial respiration and turnover and reduced de novo lipogenesis. As hepatocytes lack the GLP-1 receptor, this effect of MEDI0382 is predominantly modulated via glucagon receptors. After treatment with MEDI0382 or g1437, a glucagon analog, isolated hepatocytes showed increased basal and maximal oxygen consumption, increased mitophagosome number, and a ∼6-fold induction in expression of mitochondrial biogenesis regulator PGC-1α (P ≤ 0.0001). We used phosphoproteomics to identify potential mechanisms of action of MEDI0382 and g1437 on lipogenesis inhibition. After g1437 treatment, primary murine hepatocytes exhibited increased phosphorylation on AMPK, GSK3-α, PHK-β, ChREBP, and HSL—all known PKA substrates. We also identified new glucagon-induced phosphorylations on PHK-α2, PYGL, and ChREBP that were not necessarily PKA consensus motifs, implicating involvement of kinases downstream of PKA. In obese mice, MEDI0382 given for 7 days showed time-dependent phosphorylation of the same sites on AMPK, ChREBP, ACC2, ACLY, and DGAT1. These phosphorylation events are predicted to be inhibitory for lipogenesis. To understand translational potential, we treated primary human hepatocytes with g1437 and found 12 homologous phospho sites, including those on AMPK, ACLY, and DGAT1. Thus, MEDI0382 provides unique antisteatotic benefits for obesity/T2DM comorbidities of NASH/NAFLD via enhanced mitochondrial function and novel glucagon-mediated inhibition of lipogenesis.

Disclosure
R.C. Laker: Employee; Self; MedImmune. M. Boland: Employee; Self; Gubra, MedImmune. A. Nawrocki: None. K.M. Mather: None. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. M.R. Larsen: None. J. Trevaskis: Employee; Self; Gilead Sciences, Inc., MedImmune. Stock/Shareholder; Self; AstraZeneca. C.J. Rhodes: Employee; Self; AstraZeneca, MedImmune.

Funding
AstraZeneca
1885-P: Caveolin1 Attenuates Palmitic Acid-Induced Hepatic Steatosis through Upregulating PGC1α-sirt3 Axis
Authors: LIN CHUWEN , ZENG WEN , LIN SHUO , LIU KUNYING , ZENG LONGYI 
Published: June 2019

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. Caveolae are flask-shaped invaginations of the plasma membrane enriched with cholesterol and phospholipid. Caveolin1 is the resident structural protein of caveolae and has previously been reported to involve in hepatic steatosis and NAFLD, while the mechanism remains elusive. Here we examined the effects of caveolin1 on palmitic acid (PA)-induced hepatic steatosis. Stable caveolin1 overexpressing HepG2 cells were developed by transfection with lentiviral particles containing human CAV1 protein (Lenti-CAV1). Lenti-Control served as a negative control. Compared with BSA treatment, PA treatment of HepG2 cells for 24 hours increased the lipid droplets and TG level (P&lt;0.01). Compared with negative control, overexpression of caveolin1 attenuated the accumulation of lipid droplets and triglyceride content in HepG2 cells (P&lt;0.05). At the same time, quantitative real-time PCR revealed that the mRNA expression of PGC1α and sirt3 were increased in caveolin1 overexpressing cells (P&lt;0.05).
In conclusion, caveolin1 may improve PA-induced hepatic steatosis through up-regulating PGC1α-sirt3 axis.

Disclosure
C. Lin: None. W. Zeng: None. S. Lin: None. K. Liu: None. L. Zeng: None.
1886-P: Hepatic Type 2 Innate Lymphoid Cells Suppress Gluconeogenesis via the IL-33/GATA3/IL-13 Axis
Authors: FUJIMOTO MASANORI , YOKOTE KOUTARO , TANAKA TOMOAKI 
Published: June 2019

Type 2 innate lymphoid cells (ILC2s) were recently identified as essential components of innate immunity. Besides, beneficial roles of ILC2s in metabolic diseases are attracting attentions. Although the liver contains various immune cells including T cells and ILC2s, little is known about ILC2s in hepatic gluconeogenesis. We therefore investigated the role of ILC2s and the effect of IL-33, a potent ILC2 activator, on hepatic gluconeogenesis in mice. We found that recombinant IL-33 injection decreased fasting blood glucose levels in wild type mice. This effect was not observed in NOD/SCID Il2rg null (NSG) mice lacking certain immune cells including ILC2s, whereas the similar response was seen in another immune deficient mice Foxnnu/nu (nude mice), which have normal development in ILC2s. IL-33 treatment also suppressed hepatic gluconeogenesis assessed by pyruvate tolerance test in wt mice, while there was no response in NSG. Moreover, ILC2 transplantation was able to rescue the absence of glucose response by IL-33 in NSG. We observed a marked increase of IL-13+ ILC2 population in liver after IL-33 treatment, and Il13-/- mice did not show the decrease of blood glucose or the suppression of gluconeogenesis by IL-33 treatment. Additionally, the transfer of Il13-/- ILC2 failed to gain IL-33 induced glucose response in NSG, suggesting that the hypoglycemic effect of IL-33/ILC2s depends on IL-13. In consistent with these results, the differentially expressed gene analysis revealed that IL-13 treatment mostly altered the gene expressions of glucose metabolic process in primary hepatocytes. Notably, we identified GATA3 as a direct regulator of the effecter molecules such as Il5, Il13 and I1rl1 in hepatic ILC2s. These phenomena were also reproducible in human ILC2s and primary hepatocyte.
In conclusion, these findings indicate that hepatic ILC2s suppress hepatic gluconeogenesis via the IL-33/GATA3/IL-13 axis, thus reducing blood glucose levels.

Disclosure
M. Fujimoto: None. K. Yokote: None. T. Tanaka: None.
1888-P: Impaired Insulin Clearance Relates to Increased Liver Fat Content in Recent-Onset Type 2 Diabetes and to Impaired Glucose Control in Recent-Onset Type 1 Diabetes
Authors: ANTONIOU SOFIA , ZAHARIA OANA P. , STRASSBURGER KLAUS , KARUSHEVA YANISLAVA , BODIS KALMAN , KUPRIYANOVA YULIYA , BURKART VOLKER , MUESSIG KARSTEN , GASTALDELLI AMALIA , RODEN MICHAEL , SZENDROEDI JULIA 
Published: June 2019

Hepatic insulin metabolism in patients with newly diagnosed diabetes is not yet understood. Insulin clearance seems to be decreased in patients with obesity, visceral adiposity and type 2 diabetes (T2D), but data compared to glucose tolerant humans have been inconclusive. To this end, persons with type 1 diabetes (T1D; n=124) or type 2 diabetes (T2D; n=401) and glucose-tolerant humans (CON; n=245) underwent hyperinsulinemic-euglycemic clamps to assess insulin sensitivity (IS) and whole-body insulin clearance (ICWBIC, ml x kg-1 x min-1). Hepatic insulin clearance was also calculated from the ratio of the areas under the curve of plasma C-peptide and insulin (0-60 min) during intravenous glucose-tolerance test (IVGTT, 0-60 min) and mixed meal tolerance test (MMTT, 0-180 min). Hepatocellular lipid content (HCL) was measured by 1H-magnetic resonance spectroscopy. Analyses were adjusted for age, sex and BMI. In T1D, ICIVGTT (1.71±1.1 vs. 2.3±0.3 and 3.2±0.3, all p&lt;0.05) as well as ICMMT (1.6±0.7 vs. 2.0±0.4 and 2.1±0.3, all p&lt;0.05) were lowest compared to T2D and CON respectively and correlated negatively with HbA1c (r=-0.242 and r=-0.279, both p&lt;0.05). In T2D, ICIVGTT was positively correlated with HbA1c (r=0.178, p&lt;0.05) as well as with IS (r=0.242, p&lt;0.05). T2D patients with hepatic steatosis (n=73) had lower ICIVGTT, ICWBIC as well as ICMMT (2.2±0.31 vs. 2.4±0.31, 1.4±0.4 vs. 1.5±0.4 and 1.9 ±0.3 vs. 2.1±0.2 respectively, (i.e., differences of 8%, 9% and 7%, all p&lt;0.05) compared to T2D without steatosis (n=53). CON with steatosis also had lower ICIVGTT (n=103, 2.1±0.2 vs. 2.3±0.35, p&lt;0.05). ICMMT positively correlated with IS (r=0.338 and r=0.187, both p&lt;0.05) in T1D and T2D, but not in CON.
In conclusion, glycemic control likely impairs insulin clearance particularly in T1D patients, whereas steatosis affects insulin clearance in T2D and in glucose tolerant humans.

Disclosure
S. Antoniou: None. O.P. Zaharia: None. K. Strassburger: None. Y. Karusheva: None. K. Bodis: None. Y. Kupriyanova: None. V. Burkart: None. K. Muessig: None. A. Gastaldelli: Consultant; Self; A. Menarini Diagnostics, Eli Lilly and Company, Genentech, Inc., Gilead Sciences, Inc., Inventiva Pharma. M. Roden: Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Poxel SA, Servier. Board Member; Self; Eli Lilly and Company. Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Sanofi. Speaker's Bureau; Self; Novo Nordisk Inc. J. Szendroedi: None.
1889-P: Inhibition of Acid Sphingomyelinase Attenuates Nonalcoholic Steatohepatitis and Atherosclerosis Induced by High-Fat Diet and LPS in LDL Receptor-Deficient Mice
Authors: LU ZHONGYANG , LI YANCHUN , LOPES-VIRELLA MARIA F. , HUANG YAN 
Published: June 2019

Macrophage-mediated inflammation plays an essential role in diabetes-associated nonalcoholic steatohepatitis (NASH) and atherosclerosis. Our previous study has shown that acid sphingomyelinase (ASMase) plays a key role in the synergistic stimulation of inflammatory signaling by saturated fatty acid palmitate and lipopolysaccharide (LPS) in macrophages. In the current study, we assessed the effect of ASMase inhibition on NASH and atherosclerosis induced by lard-based, high palmitate-containing high-fat diet (HP-HFD) in combination with LPS in LDL receptor-knockout (LDLR-/-) mice. LDLR-/- mice were fed HP-HFD for 20 weeks and treated with or without a low dose of LPS and amitriptyline, an inhibitor of ASMase. Metabolic assays showed that amitriptyline reduced bodyweight, lipids and insulin resistance induced by HP-HFD or HP-HFD plus LPS. Histological study and Oil Red O staining of livers showed that while mice fed HP-HFD without LPS treatment had hepatic steatosis and hepatocellular ballooning, LPS further increased steatosis and ballooning by 70%. However, amitriptyline attenuated the effect of HP-HFD or HP-HFD plus LPS by 66%. Immunohistochemistry study showed that while mice fed HP-HFD without LPS treatment had a marked immunostaining of F4/80, a biomarker for macrophages, LPS further enhanced F4/80 immunostaining. However, amitriptyline significantly reduced F4/80 immunostaining. Furthermore, results showed that amitriptyline also attenuated atherosclerosis induced by HP-HFD plus LPS. To elucidate the underlying mechanisms, we found that amitriptyline downregulated proinflammatory cytokines by potently reducing sphingosine 1 phosphate, a bioactive sphingolipid. Taken together, this study showed that ASMase inhibition attenuated both NASH and atherogenesis induced by HP-HFD plus LPS and one of the mechanisms potentially involved in the attenuation is inhibition of S1P production.

Disclosure
Z. Lu: None. Y. Li: None. M.F. Lopes-Virella: None. Y. Huang: None.

Funding
U.S. Department of Veterans Affairs (1I01CX001825-01); National Institutes of Health
1891-P: Histone Acetylation Was Involved in the Mechanism of Regulation of PNPLA3 Gene Expression
Authors: XU XIAO , LIANG HUA , CHEN YUNZHI 
Published: June 2019

Background: PNPLA3 is a gene regulated by nutritional status, which is closely related to non-alcoholic fatty liver disease. However, the mechanism of regulation for PNPLA3 remains to be fully clarified. The aim of this study was to investigate the acetylation level of H3K9 in the promoter region of PNPLA3 gene under different physiological and nutritional conditions, which might lay a foundation for understanding the epigenetic mechanism of PNPLA3 expression regulation. C57BL/6 mice were fed ad libitum (NC, N=10), fasted (N=10) for 24 hours, and re-fed (N=10) for 12 hours. Chromatin immunoprecipitation(CHIP)-qPCR were used in assaying the enrichment for histone active marks H3K9ac in liver PNPLA3. Real-time PCR was used to measure the mRNA expression of PNPLA3, and SIRT1 which is a known deacetylase affected by nutritional status.
Results: The results showed that the H3K9ac level in the PNPLA3 promoter was significantly decreased by fasting as compared with NC mice, while the inhibited H3K9ac level of fasting increased significantly after refeeding. The PNPLA3 mRNA level was markedly downregulated during fasting (0.90±0.16 vs. 2.03±0.55, P&lt;0.05) and upregulated about 6-fold when fasted mice were refed (6.25±0.96 vs. 0.90±0.16, P&lt;0.05). While contrary to PNPLA3, the mRNA expression of SIRT1 increased significantly during fasting (2.06±0.10 vs. 1.01±0.13, P&lt;0.05) and decreased significantly after refeeding (0.99±0.04 vs. 2.06±0.10, P&lt;0.05).
Conclusions: PNPLA3 gene expression is regulated by histone acetylation and SIRT1 may be involved in this regulatory mechanism.

Disclosure
X. Xu: None. H. Liang: None. Y. Chen: None.
1897-P: Vertical Sleeve Gastrectomy Corrects the Metabolic Dysregulation Induced by Hepatocyte-Specific Ablation of p53 in Mice
Authors: HOLTER MARLENA M. , GARIBAY DARLINE , MCGAVIGAN ANNE K. , NGUYEN LILY , MOORE ELIZABETH , DAUGHERITY ERIN , COHEN PAUL , KELLY KATHLEEN M. , WEISS ROBERT , CUMMINGS BETHANY 
Published: June 2019

p53 has been implicated in the pathogenesis of obesity and diabetes, in part, by regulating whole body energy homeostasis. Conflicting evidence from mice with global alterations in p53 expression suggests the effects of p53 on metabolism are tissue-specific. Despite the critical metabolic role of the hepatocyte, the role of hepatocyte p53 is undefined. Bariatric surgery, such as vertical sleeve gastrectomy (VSG), causes high rates of type 2 diabetes remission and improves energy homeostasis at the cellular level. To define the role of hepatocyte p53 in metabolic regulation and the impact of VSG in this context, we performed VSG or sham (S) surgery in high fat diet-fed male hepatocyte-specific p53 knockout (KO) and wild type (WT) mice (n=9-12). Sham-operated mice were fed ad libitum (S-AL) or food restricted to match their body weight to VSG-operated mice (S-WM). Body weight, food intake, adiposity, energy expenditure, and oral glucose tolerance were measured. Hepatocyte p53 ablation increased body weight which was corrected by food restriction and VSG (final weight (g): S-AL-WT=33±1, S-WM-WT=27±1, VSG-WT=29±1, S-AL-KO=38±1, S-WM-KO=28±1, VSG-KO=28±1, P&lt;0.05). Hepatocyte p53 ablation decreased energy expenditure which was corrected by VSG but not by food restriction (EE (kcal/kg/min x 24hr): S-AL-WT=452±19, S-WM-WT=428±13, VSG-WT=527±37, S-AL-KO=396±10, S-WM-KO=422±10, VSG-KO=533±31, P&lt;0.05). Hepatocyte p53 ablation impaired glucose homeostasis which was improved by food restriction and to a greater extent by VSG (glucose iAUC (mg/dl x 120min): S-AL-WT=17390±2736, S-WM-WT=16191±742, VSG-WT=8769±1214, S-AL-KO=21975±1841, S-WM-KO=14764±1190, VSG-KO=8832±867, P&lt;0.05). These data reveal hepatocyte p53 is a key regulator of body weight and glucose homeostasis, largely through regulation of energy expenditure. VSG overcomes energetic impairments induced by hepatocyte p53 ablation by improving energy expenditure and glucose metabolism.

Disclosure
M.M. Holter: None. D. Garibay: None. A.K. McGavigan: None. L. Nguyen: None. E. Moore: None. E. Daugherity: None. P. Cohen: None. K.M. Kelly: None. R. Weiss: None. B. Cummings: None.

Funding
Cornell University; National Cancer Institute (R21CA195002)
1900-P: Very Low-Density Lipoprotein Ceramides and Hepatic Lipid Accumulation
Authors: MUCINSKI JUSTINE M. 
Published: June 2019

Ceramides (CER) have been implicated, along with other lipotoxic lipids (diacylglycerols, triacylglycerols, TG) that play important roles in the development and progression of nonalcoholic fatty liver disease (NAFLD). No study to date has measured CER in very low-density lipoproteins (VLDL), along with other lipids from obese men with characteristics of the metabolic syndrome. Twelve subjects with metabolic abnormalities that put them at risk for NAFLD (mean±SD, age 42.1±5.4y, BMI 37.8±3.5 kg/m2, ALT 45±21 U/L) were studied. VLDL particles were isolated in the fasted state and CER measured via UPLC-MS/MS analysis. VLDL-apoB100 was measured by ELISA and intrahepatic triglycerides (IHTG) analyzed by MRS. Total VLDL-CER were positively correlated with both VLDL-TG (r = 0.572, P=0.004) and VLDL-apoB100 (r = 0.557, P=0.005). CER were negatively correlated with IHTG (r = -0.447, P= 0.02), as was VLDL-TG concentration (r = -0.551, P=0.006). These data suggest that CER and TG are packaged together in the liver and secreted on VLDL particles, and do not support the addition of CER to particles after they have been secreted. In these relatively healthy men, the data support the concept that liver lipid secretion is associated with a decreased risk for fat accumulation in the liver. An excretion of lipotoxic lipids like TG and CER in VLDL can protect the liver from excess lipid accumulation.

Disclosure
J.M. Mucinski: None.
1916-P: Effects of Dietary Iron and Hypoxia on Glucose Homeostasis in a Fast Food-Diet Mouse Model
Authors: HARRISON ALEXANDRIA V. , JAIN SHALINI , LORENZO FELIPE , MCCLAIN DON 
Published: June 2019

Excess intake of the micronutrient iron has been associated with increased risk of type 2 diabetes (T2D). Sentinel abnormalities observed in T2D are reduced insulin sensitivity, impaired insulin secretion, and increased hepatic glucose production. All three hallmarks are significantly affected by tissue iron levels, with these effects in part being exerted through the iron-dependent hypoxia sensing pathway. The interplay between iron and hypoxia in the progression of T2D has not been thoroughly investigated. We therefore fed mice a fast food (FF) diet with varying iron concentrations, from a low 4 mg/kg to a high 2000 mg/kg, under normoxic and hypoxic conditions. There was a reduction in weight for hypoxic mice (by 23% for 4mg-Fe diet, 14% for 35mg-Fe diet, and 10% for 2000mg-Fe diet; p&lt;0.05) as compared to normoxic mice, independent of food consumption. Although higher iron was generally associated with worsening of fasting glucose (FG) and glucose tolerance for both normoxic and hypoxic mice, these parameters were significantly improved under hypoxia (10-40% improvement for FG, p&lt;0.05 and 24-36% improvement for glucose tolerance, p&lt;0.01). High iron decreased the homeostasis model assessment of insulin resistance (HOMA-IR) in normoxic mice, and hypoxic mice were less insulin resistant than those in normoxia (p&lt;0.05). Higher iron also decreased the homeostasis model assessment of beta-cell function (HOMA-B) for both normoxic and hypoxic mice, and hypoxic mice trended to have higher HOMA-B. Hypoxia-inducible factor 1-alpha (HIF1α) protein is stabilized under hypoxia, and expression increases with iron. We conclude that the effects of dietary iron to alter glucose homeostasis are, in part, dependent on oxygen levels. Both iron and hypoxia affect glucose tolerance in mice on a FF diet, and their synergistic effects may be mediated by the two regulatory arms of the hypoxia inducible factor (HIF) pathway, iron-dependent dioxygenases prolyl-hydroxylase (PHD) and factor inhibiting HIF (FIH).

Disclosure
A.V. Harrison: None. S. Jain: None. F. Lorenzo: None. D. McClain: None.

Funding
U.S. Department of Veterans Affairs; National Institute of Diabetes and Digestive and Kidney Diseases